Immunodiagnostic Biomarkers for Hepatocellular Carcinoma (HCC): The First Step in Detection and Treatment

Hepatocellular carcinoma (HCC) exerts huge effects on the health burden of the world because of its high mortality and poor prognosis. HCC is often clinically detected late in patients. If HCC could be detected and treated earlier, the survival rate of patients will be greatly improved. Therefore, identifying specific biomarkers is urgent and important for HCC. The liver is also recognized as an immune organ. The occurrence of HCC is related to exacerbation of immune tolerance and/or immunosurveillance escape. The host immune system plays an important role in the recognition and targeting of tumor cells in cancer immunotherapy, as can be seen from the clinical success of immune checkpoint inhibitors and chimeric antigen receptor (CAR) T cells. Thus, there is a pressing medical need to discover immunodiagnostic biomarkers specific to HCC for understanding the pathological mechanisms of HCC, especially for immunotherapy targets. We have reviewed the existing literature to summarize the immunodiagnostic markers of HCC, including autoantibodies against tumor-associated antigens (TAAs) and exosomes, to provide new insights into HCC and early detection of this deadly cancer.


Introduction
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, which constitutes around 80% of cases [1]. It poses a huge health risk and is the sixth most diagnosed cancer worldwide and the fourth leading cause in 2018. [2]. Contributing factor to its poor prognosis is the absence of obvious symptoms during the early stages of HCC. Consequently, greater than 60% of patients with HCC are diagnosed in advanced stages [3], leading to an extremely low overall five-year survival rate (less than 16%) [4]. However, there are many effective curable therapies for early HCC, making a good prognosis for early patients. For example, when a patient is diagnosed with HCC in Barcelona-based clinical liver cancer (BCLC) stage 0 and A, the 5-year survival rate is higher than 93% through surgical intervention therapies [5]. Thus, novel biomarkers for early HCC detection significantly impact curative treatment regimens. This exciting development of HCC biomarkers may encourage the use of more effective and novel chemoprevention strategies for people at high risk for HCC, such as HBV-infected individuals.
The ideal HCC biomarker is one that can be widely employed during a rapid and inexpensive screening process, and even asymptomatic patients can be diagnosed by trained clinicians. Generally, clinically useful biomarkers can achieve at least 90% sensitivity and specificity levels, and are non-invasive and cost-effective to be widely used [6]. Therefore, shown that the study of anti-TAAs panels as biomarkers acts as a potential early detection method for HCC. study, a panel of 12 TAAs was used to establish a predictive value based on the ser 160 HCC patients and 90 normal controls and then successfully used to diagnose H 16 of 17 patients who had no earlier clinical information prior to diagnosis [20]. In of studies, it was shown that the study of anti-TAAs panels as biomarkers acts as a tial early detection method for HCC. Figure 1. Using immunoproteomic approach for identification and validation of TAAs. Firstly, the sera from HCC patien with high titer fluorescently stained were selected out with western blotting analysis and indirect immunofluorescence using cultured tissue cells as antigen. Candidates of TAA were then separated using 2D-SDS-PAGE and analyzed by ma spectrometry. Finally, multiple methods, such as enzyme-linked immunosorbent assay (ELISA), Western blotting a immunohistochemistry (IHC), and tissue arrays, are used to validate identified potential tumor-associated antigen-an body systems. These validated TAAs can be used to form TAA arrays for the immunodiagnosing early-stage cancers.
The nature of autoantibodies against different types of TAAs is different. For suppressors, the most likely reason is mutation. TP53, as a famous tumor suppresso frequently mutated in HCC patients (~30%), and its autoantibodies can be detected of HCC patients. Different from this, the autoantibodies against the proteins enco oncogenes are usually produced, due to the protein overexpression [21,22]. Oncop IMP1, IMP2/p62, and IMP3/Koc belong to the IGF-II mRNA binding protein (IMP ily. They are highly conserved and share a similar structure: The identity of the amino acid sequences approximately is 60-80%; all three members have six charac RNA binding modules, including two N-terminal RNA recognition motifs (RRM four heteronuclear ribonucleoproteins (hnRNP) K-homology (KH) domains in thei minal regions [23]. IMPs are mainly expressed during embryonic development and disappear completely in adult tissues. Nevertheless, they are again highly express variety of cancer tissues, so they are considered oncofetal proteins [24,25]. In 1999, w identified IMP2 as a TAA in the sera of HCC patients. The autoantibodies agains can be detected in the early stage of HCC and approximately 21% of HCC patients In subsequent work, we found that the expression of IMP2 was elevated in both and protein levels in HCC. Overexpression of IMP2 can enhance the signal of the Catenin pathway in HCC，induce the genomic instability and improve the mi ability of HCC cells [26,27]. In addition, IMP1 and IMP3 play important roles in the opment of HCC [28,29]. We also found a high frequency of autoantibodies of IM IMP3 existed in the sera of HCC patients [19]. The IGF2 mRNA is one of the co targets of IMPs, which is a fetal growth factor and has a similar structure and func Figure 1. Using immunoproteomic approach for identification and validation of TAAs. Firstly, the sera from HCC patients with high titer fluorescently stained were selected out with western blotting analysis and indirect immunofluorescence by using cultured tissue cells as antigen. Candidates of TAA were then separated using 2D-SDS-PAGE and analyzed by mass spectrometry. Finally, multiple methods, such as enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemistry (IHC), and tissue arrays, are used to validate identified potential tumorassociated antigen-antibody systems. These validated TAAs can be used to form TAA arrays for the immunodiagnosing early-stage cancers.
The nature of autoantibodies against different types of TAAs is different. For tumor suppressors, the most likely reason is mutation. TP53, as a famous tumor suppressor gene, frequently mutated in HCC patients (~30%), and its autoantibodies can be detected in 20% of HCC patients. Different from this, the autoantibodies against the proteins encoded by oncogenes are usually produced, due to the protein overexpression [21,22]. Oncoproteins IMP1, IMP2/p62, and IMP3/Koc belong to the IGF-II mRNA binding protein (IMPs) family. They are highly conserved and share a similar structure: The identity of the overall amino acid sequences approximately is 60-80%; all three members have six characteristic RNA binding modules, including two N-terminal RNA recognition motifs (RRMs), and four heteronuclear ribonucleoproteins (hnRNP) K-homology (KH) domains in their C-terminal regions [23]. IMPs are mainly expressed during embryonic development and almost disappear completely in adult tissues. Nevertheless, they are again highly expressed in a variety of cancer tissues, so they are considered oncofetal proteins [24,25]. In 1999, we first identified IMP2 as a TAA in the sera of HCC patients. The autoantibodies against IMP2 can be detected in the early stage of HCC and approximately 21% of HCC patients [19,23]. In subsequent work, we found that the expression of IMP2 was elevated in both mRNA and protein levels in HCC. Overexpression of IMP2 can enhance the signal of the Wnt/β-Catenin pathway in HCC, induce the genomic instability and improve the migration ability of HCC cells [26,27]. In addition, IMP1 and IMP3 play important roles in the development of HCC [28,29]. We also found a high frequency of autoantibodies of IMP1 and IMP3 existed in the sera of HCC patients [19]. The IGF2 mRNA is one of the common targets of IMPs, which is a fetal growth factor and has a similar structure and function to IGF1 [30]. IGF2 is not expressed in healthy adults. However, the frequency of various cancers occurred more frequently in IGF2 transgenic mice [31]. In human HCC, overexpression of IGF2 is associated with fetal malformations and a variety of cancers [32]. Under the regulation of IMPs, the expression of IGF2 in HCC was significantly increased [33]. Meanwhile, IGF-2 can also be recognized as a cofactor or a second signal for transformation in SV40 oncogeneinduced tumorigenesis [34]. In summary, the TAAs like IMPs, can play an important role in carcinogenesis by modulating its downstream signaling, and their autoantibodies could be regarded as valuable biomarkers in the early diagnosis of HCC.

HCC-Derived Exosome
Exosomes are membrane-bound extracellular vesicles that are 40-100 nm in size. Under normal and pathological conditions, almost all cell types secrete exosomes. Exosomes existing in various body fluids (blood, urine, ascites, and et al.) cargoes a wide range of biomolecules, including lipids, messenger RNAs (mRNAs), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and proteins [82]. These features of exosomes could act as potential diagnostic and monitoring biomarkers for cancer [83]. (1) The amount and content of exosomes reflect the actual conditions of their original cells with high sensitivity, because exosomes can be immune-isolated using antibodies against tissue-specific proteins on the cell membrane surface. (2) The stable structure of lipid bilayer protects exosomal cargoes from damage by widespread RNases and proteases in circulation, thus exosomes can keep their cargos intact and have high fidelity and consistency. (3) Release of exosomes into biological fluids provides a non-invasive way to determine patients with tumors. (4) In contrast to the hardly detectable signal in biological fluids, exosomes comprise abundant specific cancer proteins, non-coding nuclear acids, and other markers, which facilitates the recognition of low abundance nucleic acids or proteins while increasing the signal-to-noise ratio during detection [84]. (5) The comparatively uncomplicated structure of exosomes reduces the complexity of the identification. Moreover, they may have circulating biomarkers with more accuracy, which can be a benefit for the early diagnosis of tumors. (6) Exosome analysis can directly generate details on disease lineage, staging, relapse, and drug reaction [85]. Therefore, exosomes might represent a "liquid biopsy" for malignancies.
Extensive researches have investigated serum exosomal miRNAs as potential biomarkers. As shown in Table 2, two exosome miRNAs, miR-21, and miR-122, appear repeatedly. Serum miR-21 is an important indicator used to independently assess relapse and is revealed to have more sensitivity than AFP in detecting HCC [86]. In comparison to normal individuals or patients with chronic hepatitis B (CHB), patients with HCC showed increased exosomal miR-21 levels, which is related to LC and advanced tumor stage. Moreover, compared with whole serum free of exosomes, the exosomal miR-21 level is obviously more elevated, which shows higher diagnostic sensitivity [87]. The above studies suggest that miRNAs in exosomes of circulation, like miR-21, can be used to predict HCC risk and detect HCC early. However, a report has also shown that miR-21 level in serum of HCC patients is decreased in comparison to chronic hepatitis patients [88]. Similarly, there is no increase in miR-21 levels of HBV-related HCC patients. Therefore, circulating miR-21 levels cannot distinguish patients with cirrhosis from those with HCC [89]. Identification techniques of miRNA, patient selection, and shortage of general internal controls of miRNA may contribute to this difference. Liver-specific miR-122 plays a suppressive role in the HCC progress by binding to target genes related to cell proliferation, migration, differentiation, apoptosis, and angiogenesis in HCC [90]. MiR-122 not only has diagnostic value for HCC, but also has been shown to have relation to the fibrosis of the liver [9,91], and viral replication rate [92]. Circulating levels of miR-122 are related to liver damage, as well as high ALT levels [93]. If the levels of exosomal miRNA match those in the parental cells, the result may suggest that exosomal miR-122 reflect residual liver function and capacity [94].
Many studies have been performed to investigate the possibility of exosomes as markers for different stages of HCC. Compared with serum circulating mi-RNA levels, serum exosomal mi-RNA levels can better distinguish HCC from CHB or LC. It has been revealed that the circulating levels of exosomal miR-18a, miR-221, miR-222, and miR-224 in HCC patients are significantly increased compared to patients of CHB or liver cirrhosis (LC) [95]. However, the serum levels of exosomal miR-101, miR-106b, miR-122, and miR-195 of HCC patients are reduced compared to CHB patients [95]. The prognosis for HCC patients remains unsatisfactory because of the great prevalence of postoperative relapse and cancer proliferation. MiR-125b in exosomes of serum is served as a biomarker in predicting relapse and survival in HCC patients after surgery [96]. In addition, exosomal lncRNA-ATB acts as a non-invasive predictor of HCC prognosis, which is independent of age, gender, the existence of LC, or cause [97]. It has been demonstrated that lncRNA-ATB promotes ZEB1 and ZEB2, thereby cause EMT, attack from cancer cells, and spread of cancer [98]. The serum level of exosomal miR-718 of recurrent HCC patients after LT is obviously reduced compared with patients free of recurrence, which has a positive relation to poor prognosis and aggressiveness of HCC patients [99]. Recently, accumulating evidence has identified that exosomes select particular miRNAs into their packages dependent on conditions [100]. The miRNA expression profiling in exosomes of the HCC cell line SMMC-7721, is different from the parental cells, suggesting that exosomes act as regulators of miRNAs in cells [101]. Exosomes can also cargo proteins with significant diagnostic or prognostic potential. Proteomics identification has found differences in serum exosomes of cholangiocarcinoma (CCA), primary sclerosing cholangitis (PSC), HCC, and normal controls. Serum exosomal galectin-3-binding protein (LG3BP) and polymeric immune receptor (PIGR) reveal more sensitivity in the detection of HCC than AFP. Both are oncogenic proteins, promoting the progression of HCC, malignant transformation, aggression, and proliferation [102].
LG3BP is also considered a biomarker for the poor prognosis of HCC [103].
Exosomes are a kind of non-invasive, sensitive, and specific biomarkers that serve as diagnostic, prognostic, and predictive markers for HCC (Table 2). Unlike conventional cancer biomarkers, including carcinoembryonic antigen and AFP, exosomal miRNAs can transfer their functions to target cells and modulate cell signaling [104]. Generally, tumor cells produce an increasing number of exosomes than normal cells [105]. Tumor-derived exosomes contribute to an appropriate microenvironment for tumor cells to proliferate, reduce in effectiveness of the drug, generate blood vessels, invasion, regulate the immune system, and form metastatic niche before metastasis [106]. In particular, exosomes are essential for cell-cell communication by transmitting information from tumor cells to nearby or distant cells. In addition, exosomes can induce immune responses and modulate the immune system [107]. Overcoming the resistance of HCC to chemotherapy is also a challenge. Exosomes display a range of HCC antigens [108]. Cancer-derived exosomes have been shown to trigger a stronger immune response mediated by dendritic cells (DC) rather than cell lysates, and ameliorate the tumor microenvironment (TEM) of HCC [109]. Exosomes secreted by AFP-expressing DCs (DEXAFP) induce intense immune responses based on antigen specificity, which lead to significantly delayed cancer development and increased survival rates in murine models of HCC. Therefore, DEXAFP may provide a promising vaccine without cells for immunotherapy of HCC [110]. Moreover, HCC cellderived exosomes enhance drug resistance of sorafenib in vitro. However, AMSC-derived exosomes are effective carriers of miRNA-122 and can increase the chemosensitivity of HCC [111]. These results provide potential immunotherapies for the improvement of therapeutic efficacy.

Other Immunodiagnostic Biomarkers
Squamous cell carcinoma antigen (SCCA) is a kind of serine protease inhibitor known as serpins [124]. Under physiological conditions, SCCA is expressed by the stratum granulosum of normal squamous epithelium. Under pathological conditions, it is found in carcinoma cells, such as lung tumors, cervical tumors, and head and neck tumors [125]. SCCA is absent in normal liver cells, whereas its expression is elevated when liver inflammation occurs [126]. Immunohistochemical results showed that variants of SCCA (SCCA1 and SCCA2) are overexpressed in biopsies specimens of HCC [124]. IgM antibodies in circulation constitute antigen-antibody complexes with specific cancer biomarkers, offers chances for the prediction and treatment of HCC patients [127]. The emergence of the biomarker IgM immune complex appears to be confirmed by a model of cancer immune editing, which recognizes natural IgMs as participants of great significance in innate immunity [128]. IgM in circulation identifies new epitopes on the surface of cancer cells and promotes the phagocytosis and clearance of transformed cells by dendritic cells and macrophages. The mechanism reveals a possible host defense that attempts to exert selective pressure on newborn neoplastic cells to fight cancer growth [129]. Compared with LC and control groups, elevated levels of SCCA-IgM immune complexes can be detected in the serum of cirrhosis and HCC patient and assessing SCCA-IgM levels have better diagnostic value than determining the corresponding single molecule [126,130,131]. A significantly positive correlation exists between SCCA-IgM and AFP [132]. The diagnostic accurateness of serum SCCA-IgM is greater than that of AFP, due to its higher AUC, sensitivity, and specificity, which distinguish HCC patients from cirrhosis patients [133]. The level of SCCA-IgM decreases after treatment, suggesting that SCCA-IgM plays a role in the prognostic prediction of HCC. Reduced levels of SCCA-IgM may relate to a decrease in the innate immunity and/or reduced release activity of tumor cells in patients tested positive. This data indicates that the reduced concentration or activity of SCCA in the liver may reflect the decreased invasion, proliferation, and anti-apoptotic properties of cancer cells [134]. Elevated serum SCCA-IgM levels of HCC patients have also been reported to have a relation to the decreased survival, suggesting that serum SCCA-IgM levels may make a significant contribution to the prediction of HCC therapy response [130,135].
In addition, an oncofetal protein, Glypican-3 (GPC3), is regarded as a potential biomarker in serum recently. It is a member of the Glypican family and is a heparan sulfate proteoglycan located on the outer surface of cell membranes [136]. GPC3 is expressed in the liver of the fetus, whereas it cannot be detected in the liver of healthy adults [137]. In contrast, GPC3 is overexpressed not only at the gene, but also protein levels in HCC patients. It has been demonstrated in a study that GPC3 mRNA is overexpressed in 55.7-100% of HCC tissues [138]. The same conclusions have also been shown in hepatitis C virus (HCV) infected HCC patients [139]. At the protein level, GPC3 is highly expressed in more than 70% of HCC patients by immunohistochemical staining (21/29), but not expressed in healthy or benign hepatic lesions, cirrhosis, hepatitis, or healthy adult tissues (0/38) [140]. More importantly, research has revealed that GPC3 levels are increased not only in liver cancer tissues, but also in serum. GPC3 can be cleaved and released from the cell surface into the serum. At the same time, GPC3 autoantibodies and GPC3-specific cytotoxic T lymphocytes (CTL) can be detected in the blood of HCC patients [141]. Therefore, the levels of serum GPC3, its autoantibodies, and even specific immune cells can be used as potential diagnostic biomarkers for HCC [142]. In liver cancer diagnosis, AFP is a traditional biomarker, and the expression of GPC3 can distinguish benign nodular liver tumor with AFP negative. Therefore, when diagnosing liver cancer, the combined use of GPC3 and AFP may make the diagnosis more reliable [143]. GPC3 has been the target of molecular imaging for early liver cancer, especially the positron emission tomography imaging technology for GPC3 has matured nowadays [144]. Various immunotherapies targeting GPC3 have also been studied, such as the use of antibodies against anti-GPC3, gene therapy against GPC3, vaccines based on GPC3 peptides, and GPC3-targeted CART therapy, etc. [145].
Chemokines in serum can also be deemed as ideal potential biomarkers for HCC diagnosis. For instance, CXCL13 can cooperate with its receptor CXCR5, attracting T helper cells and to encourage lymphocyte homing of naive B cells [146]. Hence, CXCL13 is also known as B cell attracting chemokine 1 (BCA 1) in humans [147]. Many studies have demonstrated that CXCL13 is deeply involved in the occurrence, development, and metastasis of multiple tumors [148,149]. In HCC, the expression of CXCL13 and CXCR5 are both up-regulated compared to healthy tissues, and even higher in poorly differentiated cancer tissues, which may be related to the harsh tumor microenvironment [150]. Clinical data showed that CXCL13 is elevated in the serum of more than 60% of HCC patients compared to healthy controls. As tumors become larger, the levels of CXCL13 in serum become higher and higher. Correlation analysis showed that serum CXCL13 levels are of high value for the diagnosis and prognosis of HCC [151]. Another clinical research also indicated that the combination of CXCL13 and AFP may potentially increase the sensitivity of AFP to HCC and can be used in the clinical diagnosis of HCC [152]. In the future, we believe that the above-mentioned potential biomarkers will be used in combination with AFP to have a more accurate diagnosis of early HCC.

Conclusions
The immune system of cancer patients suggests that it can perceive structure, function, internal location of cells, and other changes in cellular participants during tumorigenesis, which can be the first sign of carcinogenesis. Immunodiagnostic markers have been described as the reporters, internal outposts, and immune surveillance of the immune system. Moreover, immunodiagnostic biomarkers, even ones with low sensitivity, have the potential to serve as an important indicator for a tumor-targeted drug. This unexpected assistance by the immune system provides cancer researchers with powerful research tools to unlock important clues to our understanding of oncogenesis and ultimately lead to better cancer treatment strategies.

Conflicts of Interest:
The authors declare that they have no financial conflict of interest.