Obesity and non-alcoholic fatty liver disease (NAFLD) are contributing to the global rise in deaths from hepatocellular carcinoma (HCC). The pathogenesis of NAFLD-HCC is not well understood. The severity of hepatic steatosis, steatohepatitis and fibrosis are key pathogenic mechanisms, but animal studies suggest altered immune responses are also involved. Genetic studies have so far highlighted a major role of gene variants promoting fat deposition in the liver (PNPLA3 rs738409; TM6SF2 rs58542926). Here, we have considered single-nucleotide polymorphisms (SNPs) in candidate immunoregulatory genes (MICA rs2596542; CD44 rs187115; PDCD1 rs7421861 and rs10204525), in 594 patients with NAFLD and 391 with NAFLD-HCC, from three European centres. Associations between age, body mass index, diabetes, cirrhosis and SNPs with HCC development were explored. PNPLA3 and TM6SF2 SNPs were associated with both progression to cirrhosis and NAFLD-HCC development, while PDCD1 SNPs were specifically associated with NAFLD-HCC risk, regardless of cirrhosis. PDCD1 rs7421861 was independently associated with NAFLD-HCC development, while PDCD1 rs10204525 acquired significance after adjusting for other risks, being most notable in the smaller numbers of women with NAFLD-HCC. The study highlights the potential impact of inter individual variation in immune tolerance induction in patients with NAFLD, both in the presence and absence of cirrhosis.

The prevalence of obesity and non-alcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC) is rising, even in the absence of cirrhosis. We aimed to develop a murine model that would facilitate further understanding of NAFLD-HCC pathogenesis. A total of 144 C3H/He mice were fed either control or American lifestyle (ALIOS) diet, with or without interventions, for up to 48 weeks of age. Gross, liver histology, immunohistochemistry (IHC) and RNA-sequencing data were interpreted alongside human datasets. The ALIOS diet promoted obesity, elevated liver weight, impaired glucose tolerance, non-alcoholic fatty liver disease (NAFLD) and spontaneous HCC. Liver weight, fasting blood glucose, steatosis, lobular inflammation and lipogranulomas were associated with development of HCC, as were markers of hepatocyte proliferation and DNA damage. An antioxidant diminished cellular injury, fibrosis and DNA damage, but not lobular inflammation, lipogranulomas, proliferation and HCC development. An acquired CD44 phenotype in macrophages was associated with type 2 diabetes and NAFLD-HCC. In this diet induced NASH and HCC (DINAH) model, key features of obesity associated NAFLD-HCC have been reproduced, highlighting roles for hepatic steatosis and proliferation, with the acquisition of lobular inflammation and CD44 positive macrophages in the development of HCC—even in the absence of progressive injury and fibrosis.

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Introduction: Heparin sulfate proteoglycans in the liver tumour microenvironment (TME) are key regulators of cell signaling, modulated by Sulfatase-2 (SULF2). SULF2 overexpression occurs in hepatocellular carcinoma (HCC). Our aims were to define the nature and impact of SULF2 in the HCC TME. Methods: In liver biopsies from 60 patients with HCC, expression and localisation of SULF2 was analysed associated with clinical parameters and outcome. Functional and mechanistic impacts were assessed with immunohistochemistry (IHC), in silico using The Cancer Genome Atlas (TGCA), in primary isolated cancer activated fibroblasts, in monocultures, in 3D spheroids and in an independent cohort of 20 patients referred for sorafenib. IHC targets included αSMA, glypican-3, β-catenin, RelA-P-ser536, CD4, CD8, CD66b, CD45, CD68 and CD163. SULF2 impact of peripheral blood mononuclear cells was assessed by migration assays, with characterization of immune cells phenotype using fluorescent activated cell sorting. Results: We report that while SULF2 was expressed in tumour cells in 15% (9/60) of cases, associated with advanced tumour stage and type-2-diabetes, SULF2 was more commonly expressed in cancer associated fibroblasts (CAFs)(52%) and independently associated with shorter survival (7.2 versus 29.2 months, p=0.003). Stromal SULF2 modulated glypican-3/β-catenin signalling in-vitro, although in vivo associations suggested additional mechanisms underlying the CAF-SULF2 impact on prognosis. Stromal SULF2 was released by CAFS isolated from human HCC. It was induced by TGFβ1, promoted HCC proliferation and sorafenib resistance, with CAF-SULF2 linked to TGFβ1 and immune exhaustion in TGCA HCC patients. Autocrine activation of PDGFRβ/STAT3 signalling was evident in stromal cells, with release of the potent monocyte/macrophage chemoattractant CCL2 in vitro. In Human PBMCs, SULF2 preferentially induced migration of macrophage precursors (monocytes), inducing a phenotypic chance consistent with immune exhaustion. In human HCC tissues, CAF-SULF2 was associated with increased macrophage recruitment, with tumouroid studies showing stromal-derived SULF2 induced paracrine activation of the IKKβ/NF-κB pathway, tumour cell proliferation, invasion and sorafenib resistance. Conclusion: SULF2 derived from CAFs modulates glypican-3/β-catenin signalling, but also the HCC immune TME, associated with tumour progression and therapy resistance via activation of the TAK1/IKKβ/NF-κB pathway. It is an attractive target for combination therapies for patients with HCC.

daily recording of ECG, blood pressure, weight, and% body-water (bioimpedance), Stroop test (hepatic encephalopathy (HE) assessment), and self-reported well-being and food/fluid/alcohol intake. Data was Blue-toothed via a secure server to the CirrhoCare ® -App, which had 2-way patient-physician communication. Hepatologists evaluated daily data and facilitated interventions as required. A matched control cohort (n=20) with advanced cirrhosis was observed in parallel. Results Patient demographics: Mean age 59±10 years, 14 male, main etiology alcohol (75%); 75% Child-Pugh class B. Fifteen patients (75%) showed good compliance, (!4 readings/ week), 2 had moderate compliance (2-4/week), and 3 had poor compliance (<2/week). In a usability questionnaire scored 1-10, the median score was !9 for any given question.Mean follow-up was 10.1±2.4 weeks. Amongst Cirrho-Care ® managed patients, 1 died and 1 received a liver transplant. Eight readmissions occurred in 5 different patients: 3 due to HE, 1 to acute-kidney injury (AKI), 1 to both AKI and HE, and 3 in the same patient to rectal bleeding. The median readmission lasted 5 (IQR 3.5-11) days, and none was >14 days. Except for the acute bleeds, we identified early signs of decompensation in all cases, e.g. failed Stroop test, hypotension or reduction/gain in body fluid (weight), and facilitated 2 short hospitalizations of the 8 total readmissions.Based on early signs of decompensation, we contacted patients on 16 other occasions, guiding intervention and likely preventing further readmissions as confirmed by an independent physician panel. Two controls died during follow-up, and there were 13 readmissions in 8 patients, lasting median 7 (IQR 3-15) days with four admissions >14 days. They had 6 unplanned paracenteses compared to 1 in CirrhoCare ® -managed patients. Conclusions CirrhoCare ® 's novel, multimodal, home-monitoring in patients with advanced cirrhosis is feasible with excellent patient engagement, and prompts early diagnosis of decompensating events and their intervention; and hospital admissions are fewer and shorter in duration than in controls. We propose the application of CirrhoCare ® for assisted, specialist, community management of advanced cirrhosis.