Hepatology Basic Science

102

Therapeutic targeting of α-specific PI3K improves chemotherapy efficacy by inhibiting hepatic stellate cell activation in liver cancer

Qi Ruan^1,2, Lu Cao¹, Haotian Yang^1,3, Leslie Burke¹, Kim Bridle^1,3, Darrell Crawford^1,3 and Xiaowen Liang^1,2,3

¹Gallipoli Medical Research, Brisbane, Australia; ²Fazer Institute, Brisbane, Australia; ³School of Medicine, Brisbane, Australia

Background and Aim: Transarterial chemoembolization (TACE), administrating high dose of cisplatin, is a standard treatment for unresectable primary liver cancer. However, Liver cancer remains clinically challenging due to chemotherapy resistance, which has been associated with cancer-associated fibroblasts (CAFs). Activated hepatic stellate cells (HSCs), the main origin of CAFs in the tumour microenvironment (TME), contribute to fibrogenesis and treatment resistance. Understanding the molecular mechanisms of HSC activation in response to chemotherapy would identify potential targets to enhance treatment efficacy in liver cancer.

Methods: CAFs subpopulations and the expression of alpha-smooth muscle actin (αSMA) were analysed in human hepatocellular carcinomas (HCC) patients with or without TACE using a single cell RNA sequencing dataset and immunohistochemistry staining on tumour tissues. The effect of chemotherapeutic drugs on HSC activation was examined in vitro by mixed-cell spheroids and conditioned medium (CM) of cisplatin pretreated Huh7 and HuCCT1 cells (human HCC and intrahepatic cholangiocarcinoma (ICC) cell lines). A pFRET HSP33 plasmid was transfected in LX2 cells (human HSC line) to monitor intracellular ROS levels in different CM. RNA sequencing profiled differential gene expression in primary HSCs in Huh7 cell CM treated with or without cisplatin. In the preclinical models of HCC and ICC, activation markers of HSCs and PI3K signalling were investigated in the tumour tissues after cisplatin treatment. Finally, PI3K α-specific inhibitors, HS-173 and alpelisib, were tested to determine their effectiveness in inhibiting chemotherapy-induced HSC activation. Combination treatment of cisplatin and alpelisib was administered to the orthotopic HCC mouse model, with tumour volume, PI3K signalling, and collagen deposition assessed using western blot and Masson trichrome staining.

Results: The proportion of CAFs shifted towards the enrichment in COL1A1+ and ACTA2+ subpopulations, and increased αSMA expression was observed in tumour tissues of liver cancer patients after TACE treatment. LX2 cells were significantly activated by cisplatin-pretreated Huh7 and HuCCT1 cells through the paracrine effects. Increased ROS level was found in LX2 cells cultured in CM of Huh7 cells pretreated with cisplatin, indicating ROS mediated HSC activation. RNA sequencing revealed PI3K signalling in cisplatin induced HSC activation. PI3K signalling molecules were assessed in the tumour tissues of HCC and ICC mouse model, with significant increase in p110α and its downstream, AKT and ERK after cisplatin treatment. Moreover, targeting p110α inhibited cisplatin-induced HSC activation. Cisplatin plus alpelisib treatment significantly reduced tumour burden in the HCC orthotopic mouse, with suppressed PI3K signalling and decreased collagen deposition.

141

Chronic effect of a novel GPR119 agonist agent, ps318, on hepatic health in diet-induced obese mice

Mohan Patil¹, Dinesh Thapa¹, Leon Warne², Elena Dallerba³, Massimiliano Massi³, Rodrigo Carlessi^1,4 and Marco Falasca⁵

¹Curtin Medical School, Curtin Health Innovation Research Institute, Curtin University, Bentley, Perth, Australia; ²Little Green Pharma, West Perth, Australia; ³School of Molecular and Life Sciences, Curtin University, Perth, Australia; ⁴Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, Nedlands, 6009, Australia; ⁵Department of Medicine and Surgery, University of Parma, Parma, 43125, Italy

Background and Aim: G-protein coupled receptor-119 (GPR119) agonism has emerged as a promising therapeutic strategy for addressing non-alcoholic fatty liver disease (NAFLD). We developed a novel small-molecule GPR119 agonist, ps318, as an oleoyl-lysophosphatidylinositol (O-LPI) mimetic that potentially targets gut-located receptors for intestinal glucagon-like peptide-1 (GLP-1) secretion. The current study investigated the chronic effect of compound ps318 as monotherapy and in combination with sitagliptin on hepatic health status in high-fat diet (HFD)-induced obese mice.

Methods: Four-week-old healthy male C57BL6/J mice were fed with HFD (45% kcal fat) for 20 weeks. Randomly assigned animals (n=10) were orally treated with compound ps318 for ten consecutive weeks in a dose escalation manner to enhance the tolerability of the compound. The treatment commenced at 10 mg/kg/day dose, escalating weekly by 10 mg/kg until reaching a maximum of 90 mg/kg/day for the final two weeks. Sitagliptin was administered orally at 20 mg/kg/day, while the control group received vehicle (0.25% carboxymethyl cellulose). In the post-treatment phase, blood samples were collected in 4-hour fasted mice for biochemical analysis of plasma glucose (PG), triglycerides (TG), total cholesterol (CHO), alanine aminotransferase (ALT), and aspartate transaminase (AST). Metabolic peptide hormone levels were measured using the Milliplex® platform. Hepatic TG, CHO, and hydroxyproline levels were quantified in frozen liver samples. The histological investigations scored hepatic steatosis, inflammation, and ballooning progression in hematoxylin and eosin-stained tissue sections.

Results: Ten-week co-administration of compound ps318 with sitagliptin resulted in higher plasmatic GLP-1 levels than the HFD control arm, which led to a significant (p<0.05) reduction in PG levels. Moreover, the combination treatment significantly decreased liver weight (p<0.0001), plasma CHO (p<0.05), ALT (p<0.0001), and AST (p<0.05) levels. Hepatic TG (p<0.0001) and CHO (p<0.001) concentrations were also found to be reduced in this arm. Furthermore, the combination treatment attenuated hepatic hydroxyproline levels, a measure of hepatic fibrosis. These biochemical changes in the combination group were paralleled by a significant reduction (p<0.01) in NAFLD activity score.

213

The anti-fibrotic therapeutic potential of a proprietary microRNA-25 mimic for chronic liver disease

Glicia de Almeida and Brianna Pollock and Michael Pearen and Diem Hoang-Le and Rakesh Veedu and Grant Ramm

QIMR Berghofer Medical Research Institute, Brisbane, Australia

Background and Aim: Hepatic fibrosis is a common response to liver damage in all chronic liver diseases, posing a critical challenge in global health. Despite its prevalence, there are currently no clinically approved therapeutics to treat hepatic fibrosis. In biomarker discovery research, we previously demonstrated dysregulated expression of microRNA-25 (miR-25) in children with cystic fibrosis ± liver disease, suggesting a role in regulating the development of liver disease. We investigated the potential mechanism of action of miR-25 in liver disease and demonstrated its anti-fibrotic activity in hepatic stellate cells (HSCs). We showed that miR-25 inhibits fibrillar collagen expression by regulating the cross-talk between the Notch1 and TGFβR pathways, through targeting of Notch1 signalling activators, ADAM-17 and FKPB-14. We have now designed a novel, chemically modified miR-25 mimic (miR-25-C3), with significantly enhanced anti-fibrotic activity compared to commercially available mimics. In this study, we investigate the therapeutic efficacy of our proprietary miR-25-C3 in HSCs in vitro, as well as in the resolution of hepatic fibrosis in a murine model of chronic liver disease.

Methods: Anti-fibrotic functions of our proprietary miR-25-C3 mimic were assessed by transfecting the human HSC cell line, LX-2, for up to 72 hours. To specifically target hepatic stellate cells (HSCs) in murine livers, we validated a Vitamin A-coupled lipid nanoparticle carrier. LX-2 cells in either single- or co-culture with HepG2 hepatocytes were transfected for 30 min and 24 hours to confirm the specific uptake of miR-25-C3 by LX-2. Healthy C57Bl/6 male mice received one intravenous tail vein injection (0.75mg/kg) of our proprietary miR-25-C3 (Cy3-labelled) mimic with livers harvested at 4, 24, 48, and 72 hours after the injection to determine the duration of anti-fibrotic efficacy. In addition, we subjected mice to hepatocellular damage using thioacetamide (TAA, 300mg/kg in drinking water) for 5 weeks to induce moderate levels of liver fibrosis. The anti-fibrotic effect of miR-25-C3 was then assessed following three intravenous tail vein injections (0.75mg/kg) during the final week of TAA treatment.

Results: Our proprietary miR-25-C3 mimic showed superior downregulation for Notch1 signalling target genes ADAM-17 and FKBP-14, compared to a commercially available miR-25 mimic, with consequent marked inhibition of TGF-β type I receptor and fibrillar collagens (col1a1, col1a2, col3a1) in HSCs, in vitro. In addition, the use of vitamin A-coupled liposomes as an HSC-specific delivery vehicle specifically delivered miR-25-C3 to HSCs both in vitro and in vivo. miR-25-C3 significantly inhibited the expression of COL1A1 in TAA-treated mice, with histological fibrosis (assessed by Sirius red histochemistry and image J quantitation) significantly reduced in TAA-treated mice.

Conclusions: These data support the novel, anti-fibrotic therapeutic potential of our proprietary miR-25-C3 mimic. We have recently commenced a preclinical trial to further assess the efficacy of miR-25-C3 in suppressing the progression of advanced liver disease and reversing established advanced hepatic fibrosis/cirrhosis in murine models of chronic liver disease.

244

Cystic fibrosis related liver disease in children: A vascular or biliary disease?

Amit Saha^1,6, Michael Stormon², Peter Lewindon³, Nicole Graf^2,4, Guy Lampe⁵ and Mark Oliver⁶

¹St John of God Midland Public Hospital, Perth, Australia; ²Children's Hospital at Westmead, Sydney, Australia; ³Queensland Children's Hospital, Brisbane, Australia; ⁴Sydney University, Sydney, Australia; ⁵PAH Anatomical Pathology at Queensland Health, Brisbane, Australia; ⁶The Royal Children's Hospital, Melbourne, Melbourne, Australia

Background and Aim: Cystic fibrosis-related liver disease (CFLD) has long been thought to be secondary to CFTR dysfunction in the biliary epithelial cells causing hepatobiliary complications, eventually progressing to cirrhosis. However, in more recent studies, non-cirrhotic portal hypertension (NCPH) has been postulated as a possible alternative mechanism contributing to CFLD. A series from Texas (Wu et al.) of 17 explanted livers in children with CFLD reported that obliterative portal venopathy (OPV) and nodular regenerative hyperplasia (NRH) - the cardinal features of NCPH, was noted to be the most prominent histological feature. We undertook a multicentre retrospective cohort study of all paediatric CFLD related liver transplants in Australia with a view to corroborate this finding in our population, with a view to inform the discussion whether shunt surgery rather than liver transplant would be the most optimal intervention in this cohort.

Methods: We collected clinical information pertaining to the status of their liver disease from children with CFLD who underwent liver transplant at the 3 paediatric liver transplant centres in Australia- Melbourne, Sydney and Brisbane. Explant histopathology slides were independently reviewed by 2 experienced pathologists based at two different anatomical pathology centres.

Results: Data was collected from 18 children (including 10 females, median age 13 years) making this the largest paediatric series reported.Pretransplant, all had evidence of portal hypertension with splenomegaly and thrombocytopenia. Median bilirubin, ALT, ALP, GGT levels and PELD/MELD, aspartate aminotransferase to platelet ratio index (APRI) and fibrosis-4 (FIB-4) scores were recorded (Table 1) to ascertain the pre-transplant liver status. All explanted livers had evidence of variable degree of biliary cholestasis/cirrhosis as the predominant histopathological feature, whereas 11 out of the 18 explants (61%) also had patchy/focal areas of NCPH, mostly within the less fibrous or macronodular areas, with prominence of dilated, thin walled vessels in the fibrous septa.

288

Impact of gut microbiota on immune modulation and response to immune checkpoint inhibitors in hepatocellular carcinoma: Insights from human and preclinical models

Jayashi Rajapakse^1,2, SJ Shen^1,2, Saroj Khatiwada^1,2, Jason Soo^1,2, Jason Behary^1,2,3 and Amany Zekry^1,2,3

¹Microbiome Research Centre, UNSW, Syndey, Australia; ²St George and Sutherland Clinical Campuses, UNSW, Sydney, Australia; ³Department of Gastroenterology and Hepatology, St George Hospital, Sydney, Australia

Background and Aim: Hepatocellular carcinoma (HCC) is the 3^rd leading cause of cancer-related mortality worldwide, accounting for over 800,000 deaths annually. The emerging standard of care for patients with unresectable HCC is Immune Checkpoint Inhibitors (ICIs). However, response rates to ICIs among HCC patients can be as low as 20%, and even these patients may respond over time. An emerging area of interest in this field stems from the finding that microbiota composition differs between responders (Rs) and non-responders (NRs) to ICIs in many types of cancer, including HCC. In line with this, clinical studies in melanoma have demonstrated that modulation of the gut microbiota can reverse NR status in some patients by reinvigorating the anti-tumour immune response. While some studies in HCC patients have established a difference in the gut microbiota composition between Rs and NRs to ICIs, they have yet to assess how these changes may influence the anti-tumour immune response. Thus, we sought to analyse how differences in microbiota composition between Rs and NRs to ICIs in HCC may shape the systemic and hepatic anti-tumour immune response.

Methods: Stool samples were collected from HCC patients before initiating ICI therapy and 12 weeks into treatment. These samples underwent 16S rRNA sequencing. To investigate how microbiota compositions from Rs and NRs differentially affect the anti-tumour immune response, mice with diethylnitrosamine (DEN) and high-fat diet-induced HCC received faecal microbiota transplants (FMT) from pooled R or NR stool samples collected before ICI treatment. At 34 weeks, mice were euthanised, and lymphocytes were isolated from tumours, liver, spleen, blood, and mesenteric lymph nodes (MLN) for complete spectrum flow cytometry analysis.

Results: In patients receiving ICI, we noted that the relative abundances of several bacterial taxa differed between Rs and NRs. Here, Dialister was found to be increased in NR faeces at baseline (P=0.043), while by 12 weeks, Rs exhibited an increased abundance of Parabacteroides (P=0.0069), Butyricimonas (P=0.048) and Ruminococcus (P=0.012), while NRs had increased Roseburia compared to Rs (P=0.049). Administration of FMT prepared from the stool of these Rs and NRs before ICI therapy commencement to a preclinical model of HCC demonstrated modulation of the intratumor, hepatic and systemic immune responses. Specifically, R FMT-recipient mice exhibited increased regulatory T cells (Tregs) compared to NR FMT in the spleen (p=<0.0001) and the liver (P=0.050). R FMT mice also had increased CD4+ PD1+ T cells in the liver (p=0.014), tumours (p=0.049), spleen (p=0.028) and MLN (p=0.0002) compared to NR FMT mice. Moreover, CD8+ PD1+ T cells were increased in R FMT mice compared to NR FMT in all tissues assessed (liver: P=0.0028, tumours: P=0.023, blood: P=0.0035, spleen: P=0.0059, MLN: P=0.031). Importantly, R FMT mice also displayed increased tumour infiltration by CD8+ T cells (P=0.034) and CD8+ central memory T cells (P=0.0037).

Conclusions: Our findings indicate that differences in microbiota composition between Rs and NRs to ICIs in HCC patients can differentially modulate the systemic, intratumor, and intrahepatic immune responses critical for ICI outcomes. Before ICI treatment, the microbiome appears to induce an immune response with increased CD8 infiltration and upregulation of Tregs and PD-1, potentially promoting tolerance and homeostasis. This microbiome-mediated immune phenotype may be ideal for ICI efficacy by providing ICI-targetable/visible inhibitory cells, facilitating their abolishment and promoting anti-tumour responses. Consistent with studies in other cancers, Rs to ICIs were shown to exhibit higher levels of PD1+ T cells and central memory T cells. These results suggest that the host’s microbiome composition before ICI therapy may shape the immune landscape, influencing treatment outcomes. Work is underway to delineate the microbiome-mediated immune response after starting ICI.

316

Pancreatic stellate cells and macrophage interactions mediated by IL-4 promote alcoholic chronic pancreatitis progression

Zhihong Xu^1,2, Parvathy Rajan^1,2, Bomi Lee³, Chamini Perera^1,2, SM Zahid Hosen^1,2, Tanzila Khan², Tzipi Cohen-Hyams², Murray Killingsworth², Sohail Husain³, Ron Pirola^1,2, Jeremy Wilson^1,2, Stephen Pandol⁴ and Minoti Apte^1,2

¹University of New South Wales, Sydney, Australia; ²Ingham Institute for Applied Medical Research, Liverpool, Australia; ³Stanford University, Stanford, USA; ⁴Cedars Sinai Medical Center, Los Angeles, USA

Background and Aim: Smoking accelerates the progression of alcoholic chronic pancreatitis (ACP) as evidenced by early development of calcification and fibrosis. However, the mechanisms mediating these effects are not fully elucidated. We hypothesise that the interactions between pancreatic stellate cells (PSCs) and infiltrating macrophages mediated by PSC-secreted IL-4 potentiate pancreatic fibrogenesis. Aims: (1) To assess the effect of IL-4 receptor blocking peptide on the progression of alcoholic pancreatitis in a physiologically relevant model induced by alcohol feeding, cigarette smoke exposure and caerulein (an analogue of the pancreatic secretagogue cholecystokinin); (2) To determine whether the PSCs – macrophage interactions mediate the fibro-inflammatory response in vitro.

Methods: (1) C57BL/6 mice were alcohol-fed (A) for 14 weeks, exposed to cigarette smoke (S) from week 5, and then divided into the following groups: i) AS; ii) AS+Cer (Caerulein challenge, 50 μg/kg, 6 IP/day, twice/week for the last 5 weeks); iii) AS+BP [IL-4 Blocking Peptide (BP), 25 mg/kg, IP once/day for the last 2 weeks]; and iv) AS+CER+BP. Pancreatic injury, fibrosis and M2 macrophage infiltration were assessed.

(2) Mouse PSCs and bone-marrow-derived macrophages (M) were cultured alone or together (n=3-4) in the presence or absence of 50mM alcohol (A) and 40μg/ml cigarette smoke extract (CSE) and/or BP. PSC activation marker collagen mRNA and the macrophage M2 phenotype marker (CD206 mRNA) were assessed.

Results: 1. Pancreatic injury, collagen and CD206 expression were significantly increased in AS+Cer group compared to AS and AS+BP groups. These effects were inhibited by BP in AS+Cer+BP group (Table).

2. a) While incubation with macrophages alone or exposure to A or CSE alone in the presence of macrophages, increased collagen mRNA expression in PSCs, the greatest increase was observed in PSCs cultured with all 3 i.e. M+A+CSE. (Table lower section). b) In the presence of PSCs, macrophages expressed significantly increased CD206 mRNA expression, an effect that was abolished by BP. M: 1.00; M+PSC: 1.72+0.31*; M+PSC+BP: 0.71+0.11; *p<0.05 vs M or M+PSC+BP.

Conclusion: Caerulein challenge significantly increased pancreatic injury, collagen deposition and M2 macrophage infiltration in alcohol-fed, smoke exposed mice; these effects were inhibited by IL-4 BP treatment. In the presence of macrophages, PSCs incubated with alcohol + smoke compounds exhibited significantly increased activation. Interestingly, macrophages co-cultured with PSCs were polarised to an M2 phenotype, a transformation that was prevented in the presence of IL-4 BP. IL-4 may represent a novel therapeutic target to inhibit PSC-macrophage interactions thereby reducing the fibrosis of alcoholic pancreatitis.

Table:

366

Genomic and epigenomic profiling of circulating tumour DNA in hepatocelullar carcinoma

Lauren Andersson^1,2,3, Maxwell Bladen¹, Jerick Guinto¹, Dineika Chandrananda^1,3, Alexander Thompson^2,3, Jessica Howell^2,3 and Sarah-Jane Dawson^1,3

¹Peter MacCallum Cancer Centre, Melbourne, Australia; ²St Vincent's Hospital, Melbourne, Australia; ³University of Melbourne, Melbourne, Australia

Background and Aim: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. There is an urgent and unmet need to develop highly sensitive and specific biomarkers to improve early diagnosis, prognosis, treatment selection and ultimately overall survival in HCC. Analysis of circulating tumour DNA (ctDNA) through a ‘liquid biopsy’ approach provides a novel opportunity for non-invasive assessment of HCC dynamics in real time. Although well established in other cancer types, ctDNA is not currently used to guide clinical care in HCC. HCC progresses through a stepwise accumulation of genomic and epigenomic modifications, culminating in clonal populations of hepatocytes capable of unregulated and unlimited proliferation. Mutational assessment and patterns of epigenomic modifications enable differentiation of HCC vs non-HCC signals in liquid biopsy. In this study, we aim to develop a multi-modal approach for sensitive and specific ctDNA detection in HCC using a highly novel technology and analytical pipeline.

Methods: Plasma for cell free DNA (cfDNA) analysis was collected from patients with HCC, chronic liver disease (CLD) and healthy controls at longitudinal time points. Cell free DNA was extracted from plasma and sequencing libraries were prepared with 20ng input cfDNA using six-letter technology (Duet evoC, Biomodal), an enzymatic conversion method which allows simultaneous sequencing of DNA bases A, T, C, G and epigenomic modifications 5mC and 5hmC to single base pair resolution. Whole genome sequencing to a depth of 30X coverage was analysed to characterize features of ctDNA including mutations, copy number alterations, fragmentation profiles and epigenomic 5mC/5hmC modifications and then applied to a classical machine learning framework to develop a multi-omic classifier for HCC detection.

Results: A total of 495 plasma samples across 260 individuals with HCC, CLD and healthy controls have been collected across the study. Analysis of the first 63 samples including 30 HCCs, 25 CLD and 8 healthy controls, identified a step wise increase in copy number alterations from early to advanced stage HCC as compared to CLD and healthy controls (Figure 1). Epigenomic analysis identified 380 statistically significant differentially methylated cytosines across 5mC and 5hmC when compared to healthy controls (FDR <0.05). Several of these differentially methylated regions localized to genes involved in HCC tumorigenesis including tumour suppressor gene CKDN2A. Analysis of the total cohort is currently ongoing and extended results will be reported on performance of the multi-omic classifier for HCC detection.

392

Organisms in the oysters: A typical presentation of an uncommon pathology

Alicia Krasovec and Claudia Rogge

Wollongong Hospital, Wollongong, Australia

Case report: A 62-year-old man was brought to the emergency department with concerns from his partner regarding worsening abdominal distension and increasing lethargy and confusion. He had a medical history significant for alcohol-related cirrhosis complicated by portal hypertension with grade 1 varices but no previously noted ascites, gastrectomy for previous gastric cancer more than 10 years ago, and previous portal vein thrombus and pulmonary embolism but no longer on anticoagulation due to recurrent bleeding from the gastric anastomosis. On examination, he was mildly hypotensive to 99/75mmHg, and tachycardic with a heart rate of 135 bpm. He was also tachypnoeic with a respiratory rate of 36 and oxygen saturations of 98% on room air. He was cachectic, with Grade III hepatic encephalopathy, and tense clinical ascites with significant generalised abdominal tenderness. Initial blood tests revealed raised inflammatory markers, with a white cell count (WCC) of 18.7x10⁹/L, and a C-reactive protein (CRP) of 69. A septic screen was completed including a chest x-ray which did not identify any new consolidation, a urine m/c/s which showed only 10-100x10⁶/L white cells and had no organism growth, 1 set of peripheral blood cultures and a diagnostic abdominal paracentesis which was sent for both m/c/s and in culture bottles. He was commenced on empiric intravenous (IV) ceftriaxone 2g daily to treat for suspected spontaneous bacterial peritonitis (SBP). Within 12 hours of collection, both the peripheral blood cultures and the ascitic fluid cultures flagged growth of a gram negative bacilli, which was subsequently identified as Shewanella algae. Ceftriaxone was continued for 14 days with the addition of IV metronidazole. Due to the severity of this patient’s illness (initial CLIF-SOFA score = 10, which was not calculated on admission) and a further deterioration in the emergency department with escalating oxygen requirements and worsening hypotension, he was admitted to the intensive care unit for further organ support. A new non-occlusive portal vein thrombus (PVT) was identified during his admission, and his admission was further complicated by a PEA arrest, fluid overload, acute COVID illness, gastrointestinal bleeding while trialled on anticoagulation, and the development of a small bowel to abdominal wall fistala which was managed conservatively owing to his general state. At the time of writing, he has recovered from his acute illnesses and is awaiting transfer to the Rehabilitation ward.

Discussion: Monomicrobial non-neturocytic bacterascites (MNB) is not a rare phenomenon, but is uncommon, often seen in the asymptomatic patient, and is thought to represent the colonisation phase of the potential progression to SBP.¹ It is defined as positive ascitic fluid cultures without ascitic fluid neutrophil count meeting diagnostic criteria for SBP, that being a polymorphonuclear leucocyte (PMN) count <250 cells/mm³.² As in SBP, gram negative bacteria dominate, however most cases are self limited (62% of cases thought to spontaneously resolve) and the use of antibiotics are not always required, unless the patient is symptomatic or febrile. Another variant of SBP with nondiagnostic PMN count is polymicrobial bacterascites, which is mostly seen in the setting of a traumatic abdominal paracentesis. SBP is seen most often in advanced cirrhosis, and occurs owing to a disturbance in the gut flora including a predisposition in these patients to bacterial overgrowth, and likely contributed to by increased intestinal permeability.³ It is also a documented phenomenon for bacteraemic seeding causing SBP from other sources of infection eg urinary tract infections, pneumococcal sepsis.

Shewanella algae is an uncommon cause of human infection with a small but increasing number of case reports documenting patient experiences, and only minimal information regarding its role in SBP including once report of a patient who died despite early antimicrobial therapy.⁴ It is a gram-negative bacillus typically found in marine environments, with pathogenic infection noted mainly in ingestion of raw seafood particularly in the immunocompromised host. On further questioning, the patient’s partner described one of the patient’s favourite foods being oysters, which he would usually eat on a weekly basis. She told of his deterioration over recent weeks, to the point where oysters were one of the only things he could tolerate as they were cold and soft. Once more alert, the patient denied any injuries to his hand sustained by the oyster shells, and examination was consistent with this. It is difficult to determine in this patient if he had developed a systemic infection with initial bacteraemia that led to his becoming unwell with decompensation of his cirrhosis, with bacteraemic seeding of the organism, or the organism had colonised the ascitic fluid and subsequently disseminated to the bloodstream via intestinal lymphatic drainage. It is unlikely in this patient, given his presentation, that antibiotics would have been ceased on the basis of his clinical presentation with no obvious source of sepsis and non-neutrocytic ascitic fluid sample, however this case does highlight the need to consider this diagnosis in a presentation of an unwell patient with cirrhosis and ascites.

References

1. Runyon, B. Spontaneous bacterial peritonitis variants, UpToDate, April 2024, https://uptodate.com.acs.hcn.com.au/contents/spontaneous-bacterial-peritonitisvariants?search=non-neutrocytic%20bacterascites&source=search_result&selectedTitle=1~150&usage_type=default&acc=36422#H5.

2. Neto, MBB, Chedid, V, Berbari, E. Monomicrobial non-neutrocytic bacterascites due to Clostridium: to treat or not to treat? [2263]. Am J Gastroenterol 2017; 112: S1244.

3. Runyon, B. Pathogenesis of spontaneous bacterial peritonitis, UpToDate, August 2023, https://www.uptodate.com.acs.hcn.com.au/contents/pathogenesis-of-spontaneousbacterial-peritonitis?search=spontaneous%20bacterial%20peritonitis&source=search_result&selectedTitle=3%7E72&usage_type=default&display_rank=3.

4. Kim, BK, Cho, SY, Kang, B, et al. A case of spontaneous bacterial peritonitis with bacteremia caused by Shewanella algae. Infect Chemother 2014; 46(4): 264-8.

421

Identification and characterisation of novel iron regulatory molecules

V. Nathan Subramaniam^1,4, Ryan Atkins^1,4, Gautam Rishi^1,4, Heidi Sutherland^2,4 and Daniel Wallace^3,4

¹Hepatogenomics Research Group, The Queensland University of Technology (QUT), Brisbane, Australia; ²Genomics Research Centre, The Queensland University of Technology (QUT), Brisbane, Australia; ³Metallogenomics Laboratory, The Queensland University of Technology (QUT), Brisbane, Australia; ⁴Centre for Genomics and Personalised Health, School of Biomedical Sciences, Queensland University of Technology (QUT), Brisbane, Australia

Background and Aim: Dysregulation of iron metabolism is associated with many clinical disorders resulting from both iron overload and deficiency. Central to iron regulation is the interaction between hepcidin, the iron regulatory hormone, and ferroportin (FPN), the sole iron exporter. Mutations in these proteins are also associated with the iron overload disorder, hereditary haemochromatosis. The current procedures used to treat iron overload such as phlebotomy or iron chelation are either invasive or result in side-effects. Identifying potential modulators of ferroportin may provide new therapeutic targets to treat iron disorders. This project was aimed at identifying novel genes which may be involved in the trafficking and regulation of ferroportin and potentially be associated with iron-related disease.

Methods: A genome-wide CRISPR/Cas9 knockout screen was performed utilising the HEK293-TRex-FPN-GFP cell line. This cell line expresses an inducible FPN-GFP fusion protein. Transfected cells were sorted based on altered GFP expression and sequenced using next generation sequencing. The MAGeCK-Flute bioinformatic analysis pipeline was applied to sequence data to identify genes that may be involved in affecting expression of ferroportin. Knockdown studies of shortlisted genes were initially performed using specifically designed siRNA to validate the original findings within the same cell model as previously conducted for the CRISPR/Cas9 screen. CRISPR/Cas9 gene editing using specific guide RNAs was then used to knockout four of these genes in a cell line (T47D) expressing endogenous ferroportin. Changes in endogenous genes were confirmed by Sanger sequencing. Alterations in FPN-GFP and endogenous cell surface ferroportin expression following siRNA or CRISPR/Cas9 knockdown were quantified using flow cytometry.

Results: Over 50 genes were found to be associated with either an increase or decrease of FPN-GFP expression with a statistical significance of P <0.01. These were further short-listed to 12 potential targets based on a variety of criteria, including relevance to iron transport or being expressed in tissues and organs relevant to iron homeostasis. Four genes implicated in affecting ferroportin expression have been validated from among the 12 shortlisted genes using the HEK293-TRex-FPN-GFP cell line and by gene-editing in T47D cells. These genes are involved in GO biological process pathways relevant to ferroportin regulation such as negative regulation of translation, protein polyubiquitination and vesicle-mediated transport.

Conclusion: Our studies have identified and characterised novel regulators of the critical iron transporter, ferroportin. Future studies of these proteins and their analysis in patients with iron disorders will yield new insights into the regulation of iron metabolism.

437

Endothelial compartment expression of prostate-specific membrane antigen (PSMA) accurately discriminates hepatocellular carcinoma (HCC) from benign tissue

Nicholas Hannah^1,2,3,4, Catherine Mitchell⁴, Tony Huang⁴, Rita Busuttil⁵, Grace Kong⁴, Siddharth Sood^2,3 and Alex Boussioutas^2,4,5,6

¹The Royal Melbourne Hospital, Parkville, Australia; ²The University of Melbourne, Parkville, Australia; ³Northern Health, Epping, Australia; ⁴Peter MacCallum Cancer Centre, Melbourne, Australia; ⁵Monash University, Melbourne, Australia; ⁶Alfred Health, Melbourne, Australia

Background and Aim: PSMA is a promising imaging target in non-prostatic malignancy. Studies have demonstrated uptake of 68Ga-PSMA on position emission tomography (PET) in HCC. The diagnostic accuracy and utility of this finding remains unclear. We aimed to characterize the distribution of PSMA expression within HCC tumours and benign liver lesions to better understand the role PSMA may play in diagnosis and management of HCC.

Methods: Stored liver tissue specimens from our tertiary institution between 01/12/2017-01/03/2023 with a histological diagnosis of HCC, dysplastic nodule, hepatic adenoma or focal nodular hyperplasia (FNH) were retrieved. PSMA immunohistochemistry (IHC) was performed. An expression score where the expression intensity and the distribution were combined for scoring was developed: negative, weak, moderate, strong.

Results: The study included 29 samples, comprising 23 cases of HCC and 6 benign lesions (4 adenomas, 1 FNH, 1 dysplastic nodule). Among the HCC cases, the median age was 71 years (IQR 59 – 77), with 18 being male (78%) and 16 with cirrhosis (70%). Hepatitis B virus (HBV) was the most common cause of liver disease (n=7, 30%). Surgical resections n = 7 (30%), while n = 16 (70%) were biopsy. Within the HCC samples, PSMA staining the endothelial compartment of tumours was detected in 21 cases (91%, Fig 1), and canalicular in two, with expression score of negative in 0, weak in 2 (8%), moderate in 6 (25%), and strong in 15 (65%). Among benign liver lesions, the canalicular compartment exhibited predominant expression, with weak expression in the dysplastic nodule, moderate in the FNH, and strong in all 4 hepatic adenomas. Predominant endothelial expression was significantly higher in HCC (91%) compared to background parenchyma and benign lesions (4%), p<0.0001. Endothelial expression accurately discriminated HCC from benign tissue, with moderate/strong expression showing sensitivity 91%, specificity 84%, positive predictive value (PPV) 84%, and negative predictive value (NPV) 92%. Strong expression was associated with advanced HCC in 11 of 15 cases (73.3%) compared to 1 of 8 cases with early-stage (12.5%, p = 0.009).

499

Characterising peritoneal macrophage responses to infection in patients with cirrhotic ascites

Scott Read^1,2,3, Dishen Corey Chen^1,3, Mehdi Ramezani-Moghadam^1,2, Jeff Wang³, Devansh Shah¹, Chandra Malladi¹, Jacob George^3,4 and Golo Ahlenstiel^1,2,3

¹Western Sydney University, Blacktown, Australia; ²Blacktown Hospital, Blacktown, Australia; ³Storr Liver Centre, Westmead Institute for Medical Research, Westmead, Australia; ⁴Westmead Hospital, Westmead, Australia

Background and Aim: Ascites is a complication of liver cirrhosis and portal hypertension, characterised by the accumulation of fluid in the peritoneal cavity. Bacterial infection of the ascitic fluid, termed spontaneous bacterial peritonitis (SBP), occurs in up to 1/4 of patients per year, increasing the risk of sepsis, encephalopathy, kidney failure and death. Immune dysfunction driven by chronic liver disease contributes to the development of SBP, however the peritoneal immune response to infection is poorly characterised in this context. Peritoneal macrophages are the first responders to infection, acting as phagocytic sentinels to clear infection. Unlike neutrophils that flood the cavity in response to infection, macrophage populations exit the peritoneal fluid via multiple mechanisms that remain poorly understood in humans. This project aims to functionally and phenotypically characterise peritoneal macrophages in SBP to determine their role in combatting acute bacterial infection, initiating adaptive immune responses, and their potential role as biomarkers for SBP diagnosis.

Methods: A cohort of >140 clinically characterised patients have been collected at Westmead and Blacktown Hospitals. Single cell RNA sequencing (scRNASeq) of peritoneal immune cells has been performed using paired (SBP-/+) samples to identify changes in cell frequency and phenotype. Immune cell samples have been analysed by flow cytometry using an 18 marker panel to characterise macrophage populations in ascitic fluid and were compared to macrophage phenotypes in ascitic fluid clots and omental fat. Mass spectrometry and ELISA have been used to characterise the ascitic fluid proteome.

Results: Flow cytometry and scRNASeq identified significant compositional changes in peritoneal immune cell populations following infection. Peritoneal macrophages reduced in frequency during SBP, including immunologically relevant macrophage sub-populations expressing CCR7 and transcripts responsible for microbial recognition, killing and aggregation. Flow cytometry identified similar populations in omental fat and peritoneal clots, suggesting that key macrophage populations exit the fluid via aggregation and chemotaxis to tertiary lymphoid organs in the omental fat. Mass spectrometry identified significant changes in the ascitic fluid proteome during infection, that are consistent with changes in peritoneal immune cell composition.

Conclusion: Bacterial infection is a significant contributor to mortality in end stage liver disease. We have identified novel populations of macrophages that respond acutely to infection. Transcriptomic and proteomic data derived from this work will enable the development of novel therapies to boost the peritoneal immune response, and will additionally provide new biomarkers of infection to allow faster and more precise diagnosis of SBP.

519

Antigen specific natural killer cells hold promise as a novel cell therapy targeting hepatitis B virus

Dishen Corey Chen^1,3, Brian Gloss⁵, Jacob George^3,4, Scott Read^1,2,3 and Golo Ahlenstiel^1,2,3

¹Blacktown Clinical School, Western Sydney University, Blacktown, Australia; ²Blacktown Hospital, WSLHD, Blacktown, Blacktown, Australia; ³Storr Liver Centre, The Westmead Institute for Medical Research, The University of Sydney, Westmead, Australia; ⁴Westmead Hospital, WSLHD, Westmead, Australia; ⁵Westmead Research Hub, The Westmead Institute for Medical Research, The University of Sydney, Westmead, Australia

Background and Aim: Chronic Hepatitis B (CHB) virus infection remains a major global health issue, contributing significantly to chronic liver disease. Current therapies reduce viral load but are largely ineffective at reducing HBV surface antigen (HBsAg) secretion, a key driver of chronicity. New therapies to eliminate circulating HBsAg, termed a functional cure, are in urgent need. Natural Killer (NK) cells, a traditional member of the innate immune system, have recently been shown to possess memory and antigen-specificity, killing cells presenting HBV antigens. The mechanism by which NK cells identify HBV antigens, however, remains unclear. This study aims to define the mechanism of antigen specificity and subsequently harness mNK cells to develop a novel cell therapy to facilitate a functional cure by eliminating HBV-infected hepatocytes in vivo.

Methods: The study recruited 20 healthy HBV-vaccinated individuals and 65 CHB patients. HBV-peptide pentamers were employed to identify and expand rare populations of HBV peptide-specific mNK cells. A 28-colour flow cytometry panel was developed, with functional markers identified by SMART-Seq, for phenotyping NK cells using the CYTEK-Aurora spectral flow cytometer.

Results: NK cells from vaccinated individuals responded solely to the vaccine antigen HBsAg, while CHB patient NK cells responded to both surface and core antigens. SMART-Seq results identified 23 NK cell functional markers potentially linked to antigen-specificity. Phenotypic analysis using a 28-colour flow panel is ongoing. Using HBV-peptide pentamers, rare populations of HBsAg and HBV polymerase-recognising mNK cells were identified in vaccinated individuals and CHB patients (Figure). These mNK cells expanded up to 15-fold in response to HBV-peptide pentamer exposure. A positive correlation between HBV pentamer binding NK cells with serum ALT and HBV DNA in CHB patients supports their potential therapeutic and clinical relevance.

572

Characterising pancreatic organoids from hereditary pancreatitis patients

James Zuiani¹, Denghao Wu¹, Griffith Perkins^1,2, Chris Drogemuller^1,2 and Toby Coates^1,2

¹University of Adelaide, Adelaide, Australia; ²Royal Adelaide Hospital, Adelaide, Australia

Introduction: Hereditary pancreatitis (HP) is an inflammatory genetic condition typically caused by uncontrolled activation of trypsin within the pancreas. Animal models struggle to recapitulate this disease due to discrepancies in key HP genes such as PRSS1 and SPINK1. Pancreatic organoids provide the opportunity to better study this condition, allowing for modelling of patient specific mutations. We have grown organoids both from healthy pancreas and from PRSS1 mutant HP samples.

Methods: Pancreatic organoids were derived from samples taken from islet isolations, with HP samples originating from patients undergoing total pancreatectomy with islet auto transplantation (TPIAT). These organoids were embedded within a Matrigel matrix and cultured with a complex organoid growth medium. Phase contrast microscopy images were taken to track organoid growth over time. Organoids were further characterized via qPCR and immunohistochemistry to determine whether they maintained an acinar phenotype and gene expression relevant to HP, while a trypsin activity assay was used to quantify the active trypsin expressed by these organoids, as well as compare this across patients.

Results: Organoids were successfully grown from HP samples and displayed growth comparable to those of normal pancreas. Characterization of these organoids showed expression of acinar genes including PRSS1 and amylase, alongside the presence of ductal genes such as Krt19, indicating the beginnings of acinar to ductal metaplasia (ADM).

Conclusions: We have demonstrated one of the first instances of growing organoids from HP patient samples, displaying the expression of relevant acinar genes. These organoid models have the potential to provide a platform for deeper research into HP, including a better understanding of unique patient gene mutations and the development of new treatments.