Introduction

Hepatocellular carcinoma (HCC) is one of the most common and second most lethal liver cancer in the world (1). Unfortunately, only 18% of patients bearing HCC have a 5-year survival rate, which places it as the second most lethal cancer, preceded only pancreatic cancer (2). Therefore, a strategy that could reduce mortality from HCC is to develop an early diagnosis approach based on identifying new biomarkers. Based on a previous transcriptomic study in our lab, which revealed that annexins gene expression was modified in microdissected HCC nodules (3). Annexins A1, A2, A5, A8, and A10 belong to the A family of annexins that are Ca²⁺-regulated phospholipid-binding proteins. Members of this family can be peripherally coupled to the surface of negatively charged membranes when they are calcium-linked. This capability provides them with membrane-bound functions, such as organization, transport, and interaction with the actin cytoskeleton (4). The rat hepatocarcinogenesis model, induced with diethylnitrosamine (DEN), has proven to be highly effective in replicating the development of cirrhosis and heterogeneous multinodular HCC within 18 weeks. DEN's alkylating properties induce chronic liver damage, leading to inflammation and fibrosis that are associated with cirrhosis and HCC. This closely resembles the development of HCC in humans (5, 6).

Aims & Methods

We aimed to further investigate the expression profile of five annexin genes in a well-established animal model of hepatocarcinogenesis associated with cirrhosis and to identify potential serological markers for early HCC. The gene expression products of annexin were analyzed by RT-qPCR and western blots. Both mRNA and proteins were extracted from rat liver tissue at preneoplastic stages at 6 and 12 weeks and neoplasia at 18 and 22 weeks. Also, immunofluorescence was performed on rat liver samples and compared to HCC of clinical origin. Plasma samples were collected from the experimental model, and we used the ELISA technique to measure the serum levels of Annexin A1, A2, and A5.

Results

A gradual increase of annexin A1, A2, A5, and A10 was observed in liver of hepatocarcinogenesis-induced animals compared to normal liver both at mRNA and protein levels, being a statistically significant increase for annexin A1 and A5 since week 6 of the hepatocarcinogenesis model. In contrast, annexin A8 showed a significant decrease in protein levels. The histological location of annexin A1, A2, and A5 was in the tumor cells. The cellular location for annexin A1 and A5 observed in the cytoplasm and membrane of normal hepatocytes showed a re-localization to the nucleus of neoplastic cells. Notably, a significant increase in the plasma level of annexin A5 from week 12 was detected, with a gradual increase until week 22 in the hepatocarcinogenesis model. Moreover, the plasmatic level of annexin A5 significantly correlated (r² = 0.8203) with the hepatic Annexin A5 level. Using the TCGA database, we found that the expression of ANXA2 (HR = 1.7, p = 0.0046) and ANXA5 (HR = 1.8, p = 0.00077) was associated with poor survival in HCC patients.

Conclusion

The results suggest that the expression profile of annexin A1, A5, and A10 may serve as early tumor markers, whereas annexin A2 and A8 could be used as markers of hepatocellular carcinoma progression, given their expression changes during advanced stages of hepatocarcinogenesis. Significantly, the presence of annexin A5 in the plasma of model animals suggests its potential as a biomarker for hepatocarcinogenesis.

References

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Clinical Case Summary

For decompensated advanced chronic liver disease (dACLD) individuals, the onset of refractory ascites (RA) represents a dramatic event (6-month survival rate < 50%). In this setting, a relevant proportion of RA patients develop Hepatorenal Syndrome-Acute Kidney Injury (HRS-AKI) with limited therapeutic and survival chances.
An 81-year-old female dACLD RA patient was admitted to our department to receive large-volume paracentesis (LVP). Eradicated HCV infection, type II diabetes, and chronic obstructive pulmonary disease were her comorbidities.
The AKI onset imposed the intravenous (i.v.) human albumin (HA) (1 g/kg/die) administration, followed by the proper dosage of terlipressin (for HRS-AKI) within the first 72 hours (h), subsequently discontinued due to the persistence of anuria and the worsening of respiratory failure.
To simultaneously address uremic symptoms and facilitate ascites drainage, a Tenckhoff catheter was placed, and peritoneal dialysis (PD) started with a single icodextrin dwell, resulting in the removal of 14 L of ascites and an improvement in AKI within the subsequent 72h. After one week, she was discharged, receiving a specific PD schedule. After the readmission due to the difficulties in administering i.v. HA, we proposed administering HA via the intraperitoneal route, creating an HA-enriched PD (PD+HA) home-performable schedule by adding two vials of 100 ml of HA 20% to 800 ml of icodextrin solution (40 g in 1000 ml/die; lasting: 12 hours; frequency: every other day) (Figure 1).
The patient has been performing this PD schedule for 12 months (~0.5 L of ascites is discharged every other day before performing PD+HA dwell). During the follow-up, no PD-related complications (e.g., peritonitis) and liver-related events requiring hospitalization have been observed.
We report the first successful use of PD+HA, highlighting the cruciality of a multidisciplinary (hepatologists-nephrologists) approach in the management of dACLD-RA HRS-AKI patients.

References

1. Moreau, R.; Delègue, P.; Pessione, F.; Hillaire, S.; Durand, F.; Lebrec, D.; Valla, D.-C. Clinical Characteristics and Outcome of Patients with Cirrhosis and Refractory Ascites. Liver Int 2004, 24, 457–464, doi:10.1111/j.1478-3231.2004.0991.x.
2. Moore, K.P.; Wong, F.; Gines, P.; Bernardi, M.; Ochs, A.; Salerno, F.; Angeli, P.; Porayko, M.; Moreau, R.; Garcia-Tsao, G.; et al. The Management of Ascites in Cirrhosis: Report on the Consensus Conference of the International Ascites Club. Hepatology 2003, 38, 258–266, doi:10.1053/jhep.2003.50315.
3. Nadim, M.K.; Kellum, J.A.; Forni, L.; Francoz, C.; Asrani, S.K.; Ostermann, M.; Allegretti, A.S.; Neyra, J.A.; Olson, J.C.; Piano, S.; et al. Acute Kidney Injury in Patients with Cirrhosis: Acute Disease Quality Initiative (ADQI) and International Club of Ascites (ICA) Joint Multidisciplinary Consensus Meeting. J Hepatol 2024, 81, 163–183, doi:10.1016/j.jhep.2024.03.031
4. Brown, E.A.; Blake, P.G.; Boudville, N.; Davies, S.; de Arteaga, J.; Dong, J.; Finkelstein, F.; Foo, M.; Hurst, H.; Johnson, D.W.; et al. International Society for Peritoneal Dialysis Practice Recommendations: Prescribing High-Quality Goal-Directed Peritoneal Dialysis. Perit Dial Int 2020, 40, 244–253, doi:10.1177/0896860819895364.