Liver cancer affected more than 900,000 individuals worldwide in 2020 and the estimated incidence is expected to rise to 1.4 million individuals in 2040 (Cancer Tomorrow, 2022). Hepatocellular carcinoma (HCC) is the most common liver cancer, accounting for 75%–85% of all primary liver tumors (Sung et al., 2021). The risk of HCC is known to be increased by external factors (such as excessive alcohol consumption, viral infections, aflatoxin exposure) inducing liver inflammation and hepatic fibrosis progression; however, the impact of internal factors (beyond fatty liver disease) increasing the risk of developing HCC is still poorly studied (Forner et al., 2018). The known HCC risk factors promote liver cirrhosis (LC), which is usually a prior finding in 80% HCC patients (Llovet et al., 2021). In fact, 2%–5% of cirrhotic patients will develop HCC annually (Fateen and Ryder, 2017), so surveillance by liver ultrasound screening is performed in LC for early diagnosis of HCC (Forner et al., 2018; Reig et al., 2021). Unfortunately, curative treatments (based on surgery or liver transplant) are available in less than 60% of cases due to late diagnosis (Cadier et al., 2017). There is an absence of tissue biopsies because diagnosis of HCC in LC patients is mainly based on dynamic imaging tests, what reduces the ability to molecularly characterize the tumor (Reig et al., 2021). In addition to imaging tests, serum markers such as alpha-fetoprotein (AFP) that is linked to the evolutionary stage of the tumor, have been used for diagnosis and prognosis of HCC (Fateen and Ryder, 2017). However, the fact that 1) AFP may be elevated in chronic hepatitis without HCC (Force et al., 2022) 2) many small-sized HCCs have normal AFP levels (Carr et al., 2018), 3) 30% of HCC patients have normal AFP levels at diagnosis (Lee et al., 2019) and 4) high AFP levels were found in HCC-free individuals (Kobeisy et al., 2012), highlights the lack of sensitivity and specificity of this marker (Bialecki and di Bisceglie, 2005). Although AFP levels greater than 400 ng/ml are considered diagnostic of HCC as well as a marker of bad prognosis marker (Bialecki and di Bisceglie, 2005), it is not recommended to use AFP as a sole marker for HCC surveillance (Hanif et al., 2022) and the combination with other blood-based biomarkers is suggested to improve HCC diagnosis (Wang and Zhang, 2020).

Liquid biopsies have the potential to improve sensitivity/specificity as they are non-invasive, represent better the tumor heterogeneity and can be used to monitor disease evolution. The most widely studied type of liquid biopsy with demonstrated clinical utility are Circulating Tumor Cells or CTCs. The evaluation of CTCs in terms of occurrence, cell count and phenotypic characterization has demonstrated its prognostic value in several solid tumors, including lung (Bayarri-Lara et al., 2016), (de Miguel-Pérez et al., 2019), breast (Nadal et al., 2013), (Ye et al., 2019) and colorectal (Delgado-Ureña et al., 2018) cancers. With respect to HCC, several meta-analyses demonstrated the diagnostic and prognostic utility of detecting CTCs (Fan et al., 2015), (Sun et al., 2017), (Cui et al., 2020), although different isolation and detection technologies may undermine their clinical utility. Isolation methodologies based on EpCAM such as CellSearch^® are widely used, although elevated EpCAM expression levels were only found in metastatic HCC lesions compared to primary and vascular invaded tumor (Tsuchiya et al., 2019). Furthermore, elevated EpCAM expression levels were also linked to poor prognosis (Shimada et al., 2019). Thus, using alternative methodologies for CTC isolation, such as those based on size (Wang et al., 2018), (Qi et al., 2018) or including other biomarkers such as glypican-3 (Court et al., 2018) among others could overcome CellSearch^® limitations. However, Wang et al. (2018) reported lack of correlation between CTCs and recurrence after liver transplantation even though CTCs were detected in more than two-thirds of HCC patients. This suggests that not only improvements for CTC isolation are needed, but also that CTC characterization might be useful for diagnostic and prognostic purposes. Furthermore, presence of Circulating Epithelial Cells (CECs) in patients with pre-malignant diseases such as chronic obstructive pulmonary disease (COPD) had prognostic and diagnostic value (Romero-Palacios et al., 2019) in the context of Cancer Interception (Serrano et al., 2020), although it has been poorly studied in the context of liver cirrhosis (Chen et al., 2020). In fact, in most studies including LC patients, CTCs/CECs were not detected (Vona et al., 2004) or reported percentages were very low (Takahashi et al., 2021), possibly due to either patient selection or isolation methodology.

One liver-specific biomarker used to isolate CTCs from HCC patients is the asialoglycoprotein receptor 1 (ASGR1) (Li et al., 2014; Court et al., 2018), that represents the human lectin subunit 1 of the asialoglycoprotein receptor. ASGR1 heterodimerizes with ASGR2 (human lectin subunit 2) to produce a transmembrane protein primarily present in sinusoidal and basolateral hepatocellular membranes. The main role of ASGR1 is to bind galactosyl residues, facilitating glycoproteins turnover (Muramatsu, 2007). In fact, it was recently studied in the context of cardiovascular diseases (Xie et al., 2021; Wang et al., 2022a) demonstrating that its inhibition reduces hypercholesterolemia and atherosclerosis (Xie et al., 2021) by increasing cholesterol excretion (Wang et al., 2022a). In the context of liver cancer, the role of ASGR1 as tumor suppressor was first suggested via its interaction with LASS2 (longevity assurance homolog 2 of yeast LAG1) (Gu et al., 2016) and more recently by its association with DNA methylation (Zhu et al., 2022), confirming the prior findings of loss of ASGR1 expression in HCC tissue (Shi et al., 2013; Witzigmann et al., 2016). Another potential biomarker of HCC is miR-122-5p, a microRNA involved in multiple physiological processes in the liver that was found to suppress cell proliferation and malignant transformation of hepatocytes (Hu et al., 2012). Downregulation of miR-122-5p in HCC patients as well as HCC-derived cell lines was demonstrated together with an inverse correlation with cyclin G1 expression (Gramantieri et al., 2007) and upregulation of miR-122-5p was shown to repress the epithelial to mesenchymal transition (EMT) through the WNT/β-cadherin signaling pathway via Snail 1/2 (Jin et al., 2017). In plasma, miR-122-5p together with other four miRNAs were described as a diagnostic tool (Jin et al., 2019) and recently, its prognostic role was demonstrated when survival of HCC patients was greater in those with higher miR-122-5p expression (Wang et al., 2022b).

In this proof-of-concept study, we evaluated the potential diagnostic and prognostic role of circulating epithelial cells (CECs) using an isolation methodology based on immunomagnetic selection with cytokeratins 7/8 (CK) followed by ASGR1 and miR-122-5p characterization in a cohort of patients suffering from LC, with and without subsequent HCC.

In this study we have demonstrated the suitability of two liver-specific biomarkers (ASGR1 and miR-122-5p) for the characterization of circulating epithelial cells (CECs) from liver cirrhosis (LC) and hepatocellular carcinoma (HCC) patients as prognostic biomarkers. This proof-of-concept study demonstrates that phenotypic characterization of CECs with either ASGR1 or miR-122-5p may allow risk-stratification in patients at earlier disease stages, which is the principal basis of precision medicine.

Currently there is a lack of diagnostic and risk-stratification tools for HCC. Evaluation of circulating tumor cells (CTCs) in advanced stages is a prognostic marker for high risk of metastasis (Wang et al., 2018) and presence of CTCs after surgery or resection is a prognostic marker for PFS in HCC patients (Cui et al., 2020); however, there are some limitations of CTC evaluation, particularly in early and pre-tumoral disease stages. Lu-Nan, Qi et al. (2018) identified CTCs in more than half of the early-stage (BCLC 0-A) HCC patients suggesting it may be a remarkably useful tool for cancer interception (Serrano et al., 2020). However, there are currently only very few studies addressing identification and characterization of CECs in pre-tumoral diseases such as LC (Chen et al., 2020).

Expression of ASGR1 preferentially in the sinusoidal and basolateral hepatocellular membranes makes this protein an important biomarker for HCC. In fact, it has been explored in the context of targeted therapies for HCC (Chen et al., 2017), (Kim et al., 2019), (Nair et al., 2019). Other roles of ASGR1 such as hepatitis C virus binding allowing viral infection (Saunier et al., 2003) or metastasis promotion by interaction with lectins in the tumor microenvironment through the EGFR-ERK pathway (Ueno et al., 2011) are also described in relation with HCC induction. Interestingly, hepatocytes expressing low levels of ASGR1 were characterized as progenitor-like cells with higher levels of EGFR, β1 and α6 integrins expression (Ise et al., 2004). Furthermore, variable ASGR1 expression was observed between tumor stages. For instance, Shi et al. (2013) demonstrated that expression of ASGR1 (based on H-scores) in normal adjacent tissue was comparable to that on hepatic cirrhosis and early HCC (grade I or well differentiated) using tissue microarrays, although its expression decreased significantly with increasing tumor stage. This suggested that ASGR1 could potentially be used as an early indicator of the disease status. Likewise, Witzigmann et al. (2016) showed lower levels of ASGR1 mRNA in HCC compared to its adjacent normal tissue, as well as a reduction of mRNA according to increasing HCC stages. Furthermore, a decreased ASGR1 mRNA expression was observed in metastatic or highly proliferative tumors (defined by Ki69 positivity) (Witzigmann et al., 2016) and overexpression of ASGR1 was shown to inhibit cell migration and invasivity potential both in vitro and in vivo (Gu et al., 2016), suppressing metastasis and serving a prognostic biomarker.

Together with ASGR, miR-122-5p is another liver-specific biomarker which tumor suppressor role has also been suggested in HCC (Gramantieri et al., 2007; Jin et al., 2017). In fact, miR-122-5p is one of the best diagnostic markers for HCC being usually elevated in circulation of HCC patients (Jin et al., 2019); however, its overexpression in circulation was not correlated with that in tissue, which levels were found to be downregulated in HCC (Gramantieri et al., 2007). Intra-tumor heterogeneity following microenvironment stress cannot be characterized by cytokeratin positive CEC counts alone. Thus, phenotypic characterization with tissue-specific biomarkers, such as ASGR1 or miR-122-5p, may be of great importance. In fact, we identified distinct CEC subpopulations based on ASGR1/miR-122-5p staining within and between patients. Interestingly, a perfect positive correlation was found between the two markers suggesting that both could become excellent prognostic biomarkers for HCC.

Our results show that both the presence of CECs and the absence of ASGR1 expression in CECs are risk factors of developing HCC. Furthermore, ASGR1 staining has been shown to be positively correlated with miR-122-5p expression, suggesting that CECs expressing both markers originated from hepatocytes; contrarily, absence of the two biomarkers suggests a more dedifferentiated state, potentially identifying more aggressive phenotypes. These data agree with those presented by Shi et al. (2013) and Witzigmann et al. (2016), who have shown decreased ASGR1 expression levels in HCC tumor tissues, both at the mRNA and protein level as well as works from Hu et al. (2012) who showed a decreased miR-122-5p expression in HCC compared to controls.

Despite its main expression in liver, ASGR1 was also shown to be expressed in other cell types such as colon (Fang et al., 2009) or peripheral blood mononuclear cells (PBMCs) (Harris et al., 2012), albeit at lower levels compared with hepatocytes. We have also observed some level of ASGR1 expression in PBMCs of some individuals (data not shown); however, any significance of such differences among patients needs to be further investigated. One of the described roles of ASGR1 is the clearance of platelets and other prothrombotic blood components (Sørensen et al., 2009). Therefore, its presence in PBMCs might arise because of this blood hemostasis process. In fact, we showed significant correlations between ASGR1 expression in CECs and INR (p = 0.025) as well as prothrombin activity (p = 0.006), validating the role of ASGR1 maintaining blood homeostasis. Furthermore, the percentage of ASGR1 expression in CECs decreased with Child Pugh-Turcotte stage suggesting it may be used as diagnostic and prognostic biomarker in earlier disease stages as an outstanding cancer interception tool (Serrano et al., 2020). Thus, our data highlights the clinical utility of characterizing CECs using ASGR1/miR-122-5p isolated from patients with chronic liver cirrhosis or HCC to identify potentially more aggressive phenotypes (loss of ASGR1/miR-122-5p) serving as prognostic tool. A larger clinical trial in which clinical utility of these two biomarkers is assessed, might allow to update the current clinical management guidelines for HCC. Incorporation of liquid biopsy based on CTC isolation and characterization using ASGR1 and/or miR-122-5p might become a non-invasive strategy for risk stratification, improving early identification of malignancy in liver cirrhotic patients.

We understand that the cohort size might impact on our ability to reach good diagnostic power for CECs and ASGR1 as biomarkers, although similar (Qi et al., 2018) and even smaller cohort sizes (Vona et al., 2004; Wang et al., 2018; Takahashi et al., 2021) have already been used by other authors. Also, we acknowledge that detection of miR-122-5p on CECs should have been performed in all samples positive for CECs; however due to funding constraints and difficulties of the methodology, we only assessed as a proof-of-concept this biomarker in a small cohort of patients. We are currently working to increase cohort size and to characterize CECs using a more comprehensive biomarker panel including ASGR1 and miR-122-5p simultaneously using our recently developed protocol (Ruiz-Rodríguez et al., 2021) to improve statistical power and to demonstrate the diagnostic utility of characterizing CECs for LC and HCC patients.

Circulating epithelial cells (CECs) may prove to become a very useful prognostic biomarker for the identification of individuals at risk of developing hepatocellular carcinoma (HCC). Both the presence of CECs and the lack of ASGR1/miR-122-5p expression in CECs were linked with HCC incidence and poorer disease outcomes, highlighting their potential as predictive biomarkers.