Background and aims: Among those with primary sclerosing cholangitis (PSC), perihilar CCA (pCCA) is often diagnosed at a late-stage and is a leading source of mortality. Detection of pCCA in PSC when curative action can be taken is challenging. Our aim was to create a deep learning model that analyzed magnetic resonance imaging (MRI) to detect early-stage pCCA and compare its diagnostic performance with expert radiologists. Approach and results: We conducted a multicenter, international, retrospective cohort study involving adults with large duct PSC who underwent contrast-enhanced MRI. Senior abdominal radiologists reviewed the images. All patients with pCCA had early-stage cancer and were registered for liver transplantation. We trained a 3D DenseNet-121 model, a form of deep learning, using MRI images and assessed its performance in a separate test cohort. The study included 398 patients (training cohort n=150; test cohort n=248). pCCA was present in 230 individuals (training cohort n=64; test cohort n=166). In the test cohort, the respective performances of the model compared to the radiologists were: sensitivity 87.9% versus 50.0%, p <0.001; specificity 84.1% versus 100.0%, p <0.001; area under receiving operating curve 86.0% versus 75.0%, p <0.001. Even when a mass was absent, the model had a higher sensitivity for pCCA than radiologists (91.6% vs. 50.6%, p <0.001) and maintained good specificity (84.1%). Conclusion: The 3D DenseNet-121 MRI model effectively detects early-stage pCCA in PSC patients. Compared to expert radiologists, the model missed fewer cases of cancer.

Purpose:To compare the outcomes of radiation segmentectomy for early-stage hepatocellular carcinoma (HCC) in patients with non-alcoholic fatty liver disease (NAFLD) versus hepatitis C virus (HCV). Materials and Methods:A retrospective analysis of consecutive patients with NAFLD- or HCV-related HCC treated with radiation segmentectomy from 01/2017-06/2022 was performed. Eligibility criteria included solitary tumor ≤8 cm or up to 3 HCC ≤3 cm, ECOG 0-1, and absence of vascular invasion or extrahepatic spread. Imaging best response was assessed per modified Response Evaluation Criteria in Solid Tumors. Target tumor and overall progression, time-to-progression (TTP), and overall survival (OS) were calculated. All outcomes were censored for liver transplantation (LT). Complete pathologic response (CPN) was assessed in patients who underwent LT. Results:Of 142 patients included (NAFLD: 61; HCV: 81), most had cirrhosis (NAFLD: 87%; HCV: 86%) and small tumors (median size NAFLD: 2.3 cm; HCV: 2.5 cm). Patients with NAFLD had higher BMI (p<0.001) and worse ALBI scores (p=0.003). Patients with HCV were younger (p<0.001) and had higher AFP levels (p=0.034). Median radiation dose (NAFLD: 508 Gy; HCV: 452 Gy) and specific activity (NAFLD: 700 Bq; HCV: 698 Bq) were similar between cohorts. Objective response was 100% and 97% in the NAFLD and HCV cohorts, respectively. Target tumor progression occurred in 1 (2%) NAFLD and 8 (10%) HCV patients. Target tumor TTP was not met for either cohort. Overall progression occurred in 23 (38%) NAFLD and 39 (48%) HCV patients. Overall TTP was 17.4 months (95% CI 13.5-22.2) in NAFLD and 13.5 months (95% CI 0.4-26.6) in HCV patients (p=0.86). LT was performed in 27 (44%) NAFLD and 33 (41%) HCV patients, with a CPN rate of 63% and 54%, respectively. OS was not met in the NAFLD cohort and was 53.9 months (95% CI 32.1-75.7) in the HCV cohort (p=0.15). Conclusion:Although NAFLD and HCV are associated with different mechanisms of liver injury, patients with early-stage HCC treated with radiation segmentectomy achieve comparable outcomes.

Barrett’s metaplasia-associated esophageal adenocarcinoma is one of the most rapidly increasing cancers in Western countries. Whereas early detection remains the cornerstone of prevention, chemoprevention is emerging as a complementary strategy. Carcinogenesis in Barrett’s mucosa is a multistep process in which cellular growth becomes progressively dysregulated. Fortunately, the process of carcinogenesis is a protracted one, which provides ample opportunity for intervention. In this review, we will discuss various potential chemoprevention targets and rationale behind their use to prevent Barrett’s related esophageal adenocarcinoma. We will also critically appraise the emerging preclinical and clinical literature regarding prevention of neoplasia in Barrett’s esophagus.

Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of TNF superfamily, is predominantly expressed on immune cells. Engagement of TRAIL with its cognate receptor, TRAIL-R (TR), leads to cancer cells apoptosis. Although the anti-tumor role of TRAIL-TR has been extensively studied, TRAIL agonists have had very limited anti-cancer activity in human clinical trials. TRAIL signaling can facilitate the myeloid response. However, this potential immunosuppressive function of TRAIL has not been examined extensively in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, has a dense immunosuppressive microenvironment. Accordingly, CCA provides a model to examine the potential immune regulatory function of TRAIL in cancer biology. Methods: Using syngeneic, orthotopic murine models of CCA, (PMID: 29464042), murine CCA cells (SB cells) that express both Trail and Trail receptor (Tr) were implanted into livers of WT Tr− / − and LyzcreTrf/f mice, the latter have a specific deletion of TRAIL-R on myeloid cells. Hence, in this model the host immune cells express Trail but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr− / − mice had a significant reduction in tumor burden compared to WT mice. Myeloid-derived suppressor cells (MDSCs) were significantly decreased in Tr− / − tumors compared to WT tumors. Implantation of SB cells into LyzcreTrf/f mice resulted in a marked reduction in tumor burden and attenuation of MDSC infiltration compared to Lyzcre mice. In vitro functional studies employing MDSCs from mice deficient in Tr (MDSC-Tr −/−) were carried out. Compared to WT MDSCs, coculture of MDSC-Tr− /- with SB cells resulted in a significant reduction in the proliferation and immunosuppressive function of MDSCs. Moreover, following cocultured with SB cells, immunofluorescence analysis demonstrated that MDSC-Tr− / − had a reduction in the nuclear translocation of the NFkB subunit P65 compared to WT MDSCs. These data suggest that TRAIL-TR augments MDSC proliferation via NFkB. In conclusion, we have demonstrated that Tr− / − mice have a significant reduction in CCA tumor burden and MDSC infiltration. These results indicate that TRAIL-TR facilitates tumor immune escape and progression by fostering MDSC immunosuppressive function and infiltration, in an NFkB-dependent manner. Direct targeting of TRAIL on CCA cells is a potential anti-tumor strategy. Citation Format: Emilien Loeuillard, Jingchun Yang, Dong Haidong, Gregory Gores, Sumera Ilyas. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-TRAIL-R Mediates Cholangiocarcinoma Tumor Immune Evasion by Enhancing Myeloid-Derived Suppressive Cell Population [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO032.

BACKGROUND:Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV). AIM:To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models. APPROACH AND RESULTS:In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non-tumor-bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10-fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor-associated macrophages and granulocytic or monocytic myeloid-derived suppressor cells. CONCLUSIONS:Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

463 Background: Molecularly targeted therapy is gaining traction in cholangiocarcinoma with the first FDA approval of a targeted agent in 2020, the FGFR inhibitor pemigatinib. Yet response rates and efficacy are modest, and most cholangiocarcinoma patients still lack targeted therapy options. The hippo pathway effector, YAP, is an oncogene that is activated in the majority of cholangiocarcinoma, yet attempts to develop YAP inhibitors have not yet been clinically successful. Recently, we described a novel mechanism of YAP regulation, via tyrosine phosphorylation by the Src-family kinase LCK. Therefore we hypothesized that LCK inhibition by may be therapeutic in cholangiocarcinoma through suppression of YAP activity. Methods: NTRC 0652-0 is a novel tyrosine kinase inhibitor with specificity for LCK. In vitro pharmacodynamics were defined. In cholangiocarcinoma cells, specificity for LCK inhibition was determined by mass spectrometry-based unbiased analysis of the tyrosine phosphoproteome following treatment with NTRC 0652-0 or genetic deletion of LCK. YAP phosphorylation and cotranscriptional activity, cell viability, and apoptosis were assessed. A panel of eight cholangiocarcinoma patient derived organoids (PDO) were characterized and tested for sensitivity to NTRC 0652-0. Two patient-derived xenograft (PDX) models bearing FGFR2-rearrangements were utilized for in vivo assessment of pharmacokinetics, toxicity, and efficacy. Results: NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in cholangiocarcinoma cells. NTRC 0652-0 treatment led to YAP inhibition and apoptotic cell death in cholangiocarcinoma cell lines, associated with inhibition of MCL1 expression. PDOs demonstrated variable sensitivity to NTRC 0652-0, correlated with basal YAP tyrosine phosphorylation and drug-induced suppression of YAP co-transcriptional activity. FGFR2-altered cholangiocarcinoma was also identified as a subset with enrichment of YAP target genes. Cells from an FGFR2-altered PDX were sensitive to NTRC 0652-0 despite being primarily resistant to pemigatinib. In two PDX models of FGFR2-altered cholangiocarcinoma, daily oral treatment with NTRC 0652-0 inhibited YAP tyrosine phosphorylation, and resulted in stable plasma and tumor drug levels, acceptable toxicity, and significantly decreased tumor growth. Conclusions: A novel LCK inhibitor, NTRC 0652-0, reduced YAP signaling and demonstrated preclinical efficacy in multiple patient-derived models of cholangiocarcinoma. LCK is a novel therapeutic target in cholangiocarcinoma, and YAP activation or FGFR2-alteration are potential biomarkers for response.

Intrahepatic cholangiocarcinoma (ICC) contains abundant myofibroblasts derived from hepatic stellate cells (HSCs) through an activation process mediated by TGF-β. To determine the role of programmed death-ligand 1 (PD-L1) in myofibroblastic activation of HSCs, we disrupted PD-L1 of HSCs by shRNA or anti-PD-L1 antibody. We find that PD-L1, produced by HSCs, is required for HSC activation by stabilizing TGF-β receptors I (TβRI) and II (TβRII). While the extracellular domain of PD-L1 (amino acids 19-238) targets TβRII protein to the plasma membrane and protects it from lysosomal degradation, a C-terminal 260-RLRKGR-265 motif on PD-L1 protects TβRI mRNA from degradation by the RNA exosome complex. PD-L1 is required for HSC expression of tumor-promoting factors, and targeting HSC PD-L1 by shRNA or Cre/loxP recombination suppresses HSC activation and ICC growth in mice. Thus, myofibroblast PD-L1 can modulate the tumor microenvironment and tumor growth by a mechanism independent of immune suppression.

Luis Antonio Díaz,1 Sumera I. Ilyas,2 and Juan Pablo Arab1 Autoimmune hepatitis (AIH) is a chronic liver disease (CLD) that frequently affects women of reproductive age. AIH disease activity or medications used in AIH management, such as steroids, may increase adverse effects during pregnancy. Complications of pregnancies in AIH after 20 weeks of gestation were assessed using retrospective data from the U.S. National Inpatient Sample. Wang et al. identified 18,595,345 pregnancies between 2008 and 2016, with 935 pregnancies (<0.001%) occurring in women with AIH; temporal trends remained stable during the study period. Multivariate analysis demonstrated that AIH patients had a higher risk of gestational diabetes and hypertensive complications (preeclampsia, eclampsia, or hemolysis, elevated liver biochemistries, low platelets syndrome) compared to patients with other CLD or no CLD. Moreover, AIH was also associated with preterm births. Despite the higher risk of these complications, women with AIH and their infants had favorable survival. Notably, patients with AIH did not have a higher risk of postpartum hemorrhage and maternal or perinatal death. These results suggest that many pregnancyrelated complications of AIH can be prevented or appropriately managed with couseling. (Hepat ology 2022;75:512).

Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor superfamily, is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable interest as a potential anti-cancer strategy, TR agonists have had very limited anti-cancer activity in humans. TRAIL signaling in T cells may also potentially provide an immune checkpoint function as it can inhibit T cell activation and proliferation by interfering with T cell receptor signaling. However, this potential immune checkpoint function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a malignancy of the bile ducts, provides a model to examine the potential immune checkpoint function of TRAIL. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both TRAIL and TRAIL receptor (TR) were implanted into livers of WT C57BL/6J and Tr−/− mice. Hence, in this model the host immune cells express TRAIL but not the receptor; they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to potential TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed and tumor and immune characterization (via flow cytometry) was conducted. Results: Implantation of SB cells into Tr−/− mice result in a significant reduction in tumor volumes compared to WT mice. Tumor-bearing Tr−/− mice had a significant infiltration of cytotoxic T lymphocytes (CTLs) (CD45+CD3+CD8+CD11a+) and enhanced CTL effector function. Moreover, Tr−/− mice tumors had a significant decrease in granulocytic and monocytic myeloid-derived suppressor cell (MDSCs) as well as tumor-associated macrophages (TAMs) (CD45+F4/80+CD11b+CD206+) compared to WT mice tumors. Conclusion: Using a unique syngeneic orthotopic implantation model of murine CCA, we demonstrate that CCA cells expressing TRAIL in a mouse genetically deficient for TR exhibit reduced tumor volumes enhanced CTL infiltration and function, and reduced MDSC and TAM infiltration into the tumors. These data suggest that TRAIL has a potential immune checkpoint function, and targeting TRAIL signaling with consequent augmentation of CTL function maybe a promising therapeutic approach in human cancers. Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera Ilyas. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates tumor immune evasion in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2738.

PURPOSE Cholangiocarcinomas (CCA) are a group of heterogeneous tumors arising from the biliary epithelia. Significant sequencing efforts have provided further insights into the molecular mechanisms of this disease including fibroblast growth factor receptor (FGFR) alterations, which occurs in approximately 15%-20% of intrahepatic CCAs. Herein, we describe the FGFR inhibitor (FGFRi)-associated treatment toxicity and cancer-specific outcomes from a multicenter single-institution cohort. METHODS This is a retrospective study of patients with CCA and known FGFR alterations treated with FGFRi. We describe the toxicity and efficacy in patients treated at Mayo Clinic between January 2010 and December 2020. RESULTS Our group identified 61 patients with advanced or metastatic CCA, 19 males (31%) and 42 females (69%), harboring FGFR alterations who received FGFRi. The most common grade 1 or higher adverse events for all patients included fatigue (92%), AST elevations (78%), anemia (80%), decreased platelet count (63%), and hyperphosphatemia (74%). Median progression-free survival on FGFRi was 5.8 months for all patients (95% CI, 4.9 to 9.0). Females had significantly longer progression-free survival at 6.9 months (95% CI, 5.2 to 11.8) on FGFRi compared with males at 4.9 months (95% CI, 2.8 to not estimable; P = .038). CONCLUSION FGFRi are well tolerated with clinical efficacy. With the recent approval of FGFRi by the US Food and Drug Administration and ongoing clinical trials for new FGFRi, understanding outcomes and toxicity associated with these medications is important for precision oncology.

Introduction: Cholangiocarcinomas (CCAs) are biliary epithelial tumors with rising incidence over the past 3 decades. Early diagnosis of CCAs remains a significant challenge and the majority of patients present at an advanced stage. CCAs are heterogeneous tumors and currently available standard systemic therapy options are of limited effectiveness. Immune checkpoint inhibition (ICI) has transformed cancer therapy across a spectrum of malignancies. However, the response rate to ICI has been relatively disappointing in CCAs owing to its desmoplastic tumor microenvironment (TME).Areas covered: Tumor microenvironment of CCAs consists of innate and adaptive cells, stromal cells, and extracellular components (cytokines, chemokines, exosomes, etc.). This intricate microenvironment has multiple immunosuppressive elements that promote tumor cell survival and therapeutic resistance. Accordingly, there is a need for the development of effective therapeutic strategies that target the TME. Herein, we review the components of the CCA TME, and potential therapies targeting the CCA TME.Expert opinion: CCAs are desmoplastic tumors with a dense tumor microenvironment. An enhanced understanding of the various components of the CCA TME is essential in the effort to develop novel biomarkers for patient stratification as well as combination therapeutic strategies that target the tumor plus the TME.

Immune checkpoint blockade (ICB) has revolutionized cancer therapeutics. Desmoplastic malignancies, such as cholangiocarcinoma (CCA), have an abundant tumor immune microenvironment (TIME). However, to date, ICB monotherapy in such malignancies has been ineffective. Herein, we identify tumor-associated macrophages (TAMs) as the primary source of programmed death-ligand 1 (PD-L1) in human and murine CCA. In a murine model of CCA, recruited PD-L1+ TAMs facilitated CCA progression. However, TAM blockade failed to decrease tumor progression due to a compensatory emergence of granulocytic myeloid-derived suppressor cells (G-MDSCs) that mediated immune escape by impairing T cell response. Single-cell RNA sequencing (scRNA-Seq) of murine tumor G-MDSCs highlighted a unique ApoE G-MDSC subset enriched with TAM blockade; further analysis of a human scRNA-Seq data set demonstrated the presence of a similar G-MDSC subset in human CCA. Finally, dual inhibition of TAMs and G-MDSCs potentiated ICB. In summary, our findings highlight the therapeutic potential of coupling ICB with immunotherapies targeting immunosuppressive myeloid cells in CCA.

INTRODUCTION: Reflux & being overweight are strong risk factors for esophageal adenocarcinoma (EAC).There is a trend toward Low-Carb (LC) diet use among both obese & healthy individuals.The LC diet reduces weight, improves insulin resistance, may decrease reflux & potentially decrease risk of EAC.However, we previously found that LC diet increased levels of procarcinogenic bile salt deoxycholic acid (DCA) in rats.Our AIM was to examine the effect of LC diet on procarcinogenic signaling in Barrett's mucosa & EAC development in rats with reflux.METHODS: Sprague-Dawley rats (n=65) were operated to induce gastroduodenal reflux & randomized at 22 weeks post-op (time to develop Barrett's).The effect of LC diet on procarcinogenic signaling in Barrett's was tested in a short term investigation involving 20 rats randomized (1:1) to LC or balanced (ADA) diet for 4 weeks.The effect of LC diet on development of EAC was examined in a long-term study involving 45 rats randomized (1:2) to LC or ADA diet for 20 weeks.Rats were sacrificed & esophageal tissue tested for PGE2 levels & key enzymes (cPLA2, COX-2) involved in PGE2 synthesis.Presence of Barrett's mucosa & EAC was noted.Serum IGF1 levels & animal weight was examined.RESULTS: The 4 week short-term LC diet significantly increased PGE2 levels (245±41 vs 95±22pg/mg, p=0.002), cPLA2 & COX-2, compared to ADA diet.As seen previously, LC diet increased levels of DCA.In-vitro assays showed that increasing DCA levels up-regulated cPLA2, increased COX-2 & induced PGE2 synthesis.LC diet was associated with weight loss but serum IGF1 levels were not different.Based on these results, we anticipated that up-regulation of carcinogenic signaling with LC diet would increase the rate of EAC.However, the rate of EAC was not different in long-term LC vs ADA rats (9/15, 60% vs 19/30, 63% p=1.0).The long-term LC diet did result in significant weight reduction (321±28gms vs 396±11gms, p=0.03) & lower IGF-1 levels(10±3 vs 1.2±1 ng/ml, p=0.02) compared to ADA diet.CONCLUSIONS: Short-term LC diet supplementation promotes procarcinogenic signaling involving PGE2 synthesis.However, this does not translate to an increased risk of EAC with long-term LC diet supplementation.A possible explanation for the bimodal effect of LC diet is the marked weight reduction & low IGF1 levels with long-term LC diet, which may negate the effects of procarcinogenic signaling.This is clinically relevant because most people use Low-Carb diet for short term rather than long term, which may increase their risk of EAC.Studies to examine the opposing effects of IGF-1/weight reduction & PGE2 pathway are currently being investigated.

INTRODUCTION: Combinational chemoprevention, whereby synergism can be achieved between two agents, represents an important development in the field of cancer prevention. Epidemiological studies on the development of esophageal adenocarcinoma suggest a positive association with reflux & a negative association with NSAIDs. We & others have reported the chemopreventive potential of NSAIDs. We also found that Urso, which can modify bile salt composition, possesses chemopreventive potential in-vitro. Our AIM was to examine the chemopreventive potential of low-dose Aspirin in combination with Urso. METHODS: Sprague-Dawley rats (n=100) were operated to induce gastroduodenal reflux into esophagus. 86 surviving rats were randomized to a diet containing 1% Urso (n=19), 0.3% Aspirin (n= 19), 1% Urso+0.15% Aspirin (n=19) or control diet (n=29) for 8 months. Rate of esophageal cancer, mucosal injury score & the rate of Barrett's esophagus was determined. Snapfrozen Barrett's tissue was analyzed for COX-2 expression & PGE2 levels. RESULTS: The combination treatment using Urso & low-dose Aspirin significantly reduced the rate of esophageal carcinoma (5/19=26%) compared to control (18/29=62% p<0.05). The use of combinational chemoprevention resulted in a relative cancer risk reduction of 58% (95% CI 45-69%) & number needed to treat was 3. The combinational treatment also reduced the PGE2 levels compared to control(132±24 vs 40±10pg/mg, p=0.002) . There was also a reduction in COX-2 expression in animals that received combinational treatment. Urso or Aspirin treatments alone were not associated with significant reduction in the risk of esophageal cancer. To our surprise, Urso alone increased the COX-2 expression, which may partly explain why Urso alone failed to decrease the risk of esophageal cancer, but was effective in reducing the cancer in combination with aspirin, which is a non-specific COX2 inhibitor. Additional mechanistic studies are underway. CONCLUSIONS: A combinational treatment with Urso & low-dose Aspirin appears to be a successful strategy to prevent the development of esophageal adenocarcinoma in animals. The cancer risk reduction with combinational therapy was associated with down-regulation of PGE2 synthesis pathway. Even though Urso is known to reduce the concentration of carcinogenic bile salts, the beneficial effects of this change were likely negated by increased PGE2 synthesis by Urso. One possible mechanism for the effectiveness of Urso & Aspirin combination was that the beneficial effects of Urso on bile salt modification were likely retained & its untoward effects were counteracted by Aspirin. A human study will be a reasonable approach.