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The male/female ratio of patients with hepatocellular carcinoma (HCC) is often unbalanced towards the male sex, indicating a sex predisposition for HCC development. A possible explanation may be attributed to different hormonal statuses, including the pro-inflammatory action of androgens in men and the protective effects of oestrogen against excessive inflammation in women. Although several studies have studied gene expression in patients with HCC, very few have attempted to identify features that could be distinctive between male and female patients. The present study aimed to identify distinctive signalling mechanisms between men and women that may be associated with HCC progression. The present study analysed a detailed microarray database that was obtained from the prospective study of 78 patients with HCC to study gene expression according to sex. In addition, the present study aimed to evaluate whether the differentially expressed genes were known oestrogen targets. Moreover, RNAs from the HCC cohort were evaluated for microRNA (miRNA/miR) expression, and a relationship between miRNA and gene expression according to sex was investigated. One gene, sineoculis homeobox homolog 1 (SIX1), which is known to be an oestrogen target gene, was revealed to be highly upregulated in hepatitis virus C (HCV)-positive female patients with HCC but not in HCV-positive male patients. In addition, SIX1 upregulation had a significant relationship with tumour growth speed (assessed as tumour doubling time in two CTs performed 6 weeks apart) and survival (P=0.009 and P=0.042, respectively) in female patients only. Furthermore, SIX1 upregulation was related with miR-421 and miR-9-5p only in male patients; however, in female patients, SIX1 upregulation had a direct relationship with miR-181b, miR-503-5p and miR-125b (miRNAs with potential oncogenic capacity), and an inverse correlation with miR139-5p, miR-26b, let7c-3p and let7c-5p (putatively oncosuppressive microRNAs). These data suggested a distinctive model for liver carcinogenesis in HCV-positive women, with downregulation of protective mechanisms against tumour progression and the activation of potential oncogenes, in relation to the oestrogen target gene SIX1. (IRB10/08_CE_UniRer; ClinicalTrials ID: NCT01657695).

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) are the most common primary liver cancers. Traditionally, both neoplasms are considered distinct entities due to their different background. However, the comparison of the proliferative subgroup of iCCA with the subgroup of aggressive HCCs (overexpressing transcriptomic signature (TS) (10.1136/gutjnl-2014-308483)) showed that they share 95% of the overexpressed genes and that they hold similar severe prognosis. In TS-positive aggressive HCCs, the most expressed gene is Angiopoietin 2 (ANGPT2), which is associated with increased angiogenesis and proliferation, epithelial-mesenchymal transition (EMT), and PD1/PDL1 activation. We therefore, studied the effect of ANGPT2 and of its regulator VEGF on in vitro 3D models, with the aim of evaluating putative common carcinogenic pathways shared by aggressive HCCs and iCCA. We generated spheroids from HepG2 and HuCCT-1 cell (HCC and iCCA derived cell lines respectively), while EGI-1 cells, from extrahepatic cholangiocarcinoma (eCCA), served as control. Four days after seeding, well-shaped spheroids were stimulated with either 200 ng/mL ANGPT2 or VEGF, or with 100 ng/mL of ANGPT2/VEGF mix. After 3 and 48h, migration of adherent spheroids and expression of EMT markers (E-cadherin, N-cadherin, Vimentin, Beta-catenin) were evaluated by Western blot (WB) and immunofluorescence. Unstimulated spheroids served as negative controls. In both HepG2 and HuCCT-1, but not in EGI-1, treatment with ANGPT2, VEGF or their combination, stimulated the migration of the adherent spheroids. With respect to EMT biomarkers, in HepG2 E-cadherin expression dropped significantly starting from 3h and remaining low even at 48h. Notably, E-cadherin expression by untreated spheroids was lower at 48h than at 3h. In HuCCT-1, E-cadherin expression levels were not modified by the treatments either at 3h or at 48h. N-cadherin expression levels increased, regardless of treatment, in HepG2 from 3h, while in HUCCT-1 from 48h. Vimentin expression strikingly increased after 48h stimulation in both HepG2 and HuCCT-1, but not in controls. No differences were observed in any of the cell lines in Beta-catenin expression for any treatment. Expression of EMT markers remained unchanged in EGI-1 regardless of any treatment,. Immunofluorescence analysis of the adherent spheroids revealed a marked decrease in E-cadherin expression at the periphery of the migrating cells in both HepG2 and HuCCT-1, paralleled by an increase of N-cadherin and Vimentin expression. Taken together, our results demonstrate a similar response to proangiogenic stimulation of both HCC and iCCA cells, but not of EGI-1, thus underlying a) the substantial phenotypic differences between eCCA and iCCA, and b) the pro-invasive effects of angiogenic growth factors on both HCC or iCCA cells Supported by AIRC under IG 2020 - ID. 24858 project – P.I. Villa Erica Citation Format: Fabiola Milosa, Adriana Romanzi, Gemma Marcelli, Rosina Maria Critelli, Simone Lasagni, Massimiliano Cadamuro, Luca Fabris, Erica Villa. Aggressive hepatocellular carcinoma and intrahepatic cholangiocarcinoma cell lines share similar response to proangiogenic priming [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 PO008.

Key Points Three-day change in angiopoietin-2 levels predicts COVID-19 in-hospital mortality, whereas the 10-day trend is associated with chronic lung disability. Angiopoietin-2 may play an important pathogenic role in patients with COVID-19, and it could be a target for new treatments., This study examined the association between dynamic angiopoietin-2 assessment and COVID-19 short- and long-term clinical course. We included consecutive hospitalized patients from 1 February to 31 May 2020 with laboratory-confirmed COVID-19 from 2 Italian tertiary referral centers (derivation cohort, n = 187 patients; validation cohort, n = 62 patients). Serum biomarker levels were measured by sandwich enzyme-linked immunosorbent assay. Lung tissue from 9 patients was stained for angiopoietin-2, Tie2, CD68, and CD34. Cox model was used to identify risk factors for mortality and nonresolving pulmonary condition. Area under the receiver operating characteristic curve (AUROC) was used to assess the accuracy of 3- and 10-day angiopoietin-2 for in-hospital mortality and nonresolving pulmonary condition, respectively. Three-day angiopoietin-2 increase of at least twofold from baseline was significantly associated with in-hospital mortality by multivariate analysis (hazard ratio [HR], 6.69; 95% confidence interval [CI], 1.85-24.19; P = .004) with AUROC = 0.845 (95% CI, 0.725-0.940). Ten-day angiopoietin-2 of at least twofold from baseline was instead significantly associated with nonresolving pulmonary condition by multivariate analysis (HR, 5.33; 95% CI, 1.34-11.77; P ≤ .0001) with AUROC = 0.969 (95% CI, 0.919-1.000). Patients with persistent elevation of 10-day angiopoietin-2 levels showed severe reticular interstitial thickening and fibrous changes on follow-up computed tomography scans. Angiopoietin-2 and Tie2 were diffusely colocalized in small-vessel endothelia and alveolar new vessels and macrophages. Angiopoietin-2 course is strongly associated with COVID-19 in-hospital mortality and nonresolving pulmonary condition. Angiopoietin-2 may be an early and useful predictor of COVID-19 clinical course, and it could be a relevant part of disease pathogenesis. Angiopoietin-2 blockade may be a COVID-19 treatment option., Visual Abstract