Diagnostic biomarkers in hepatocellular carcinoma

Being the second most lethal type of cancer, Hepatocellular carcinoma (HCC) annually kills 650,000 individuals worldwide, approximately, prompting communal malignant tumor that accounts for more than 90% of liver cancer. We aimed to clarify the functioning of different diagnostic markers of incidence and progression of hepatocellular carcinoma. Biomarkers are molecular indicators that are very imperative for the management of different diseases. Different biomarkers of HCC, such as serum and tumor biomarkers have been diagnosed and their presence in urine, tissues and serum indicates the development of the tumor. The diagnoses at early stages helps in the prevention of HCC. In the case of HCC, more than 70% of patients showed an increased level of alpha-fetoprotein (AFP) due to excretion of the tumor and thus the level of AFP in the body is one of the most important biomarkers of HCC. Another important biomarker is AFPL3 is considered as more precise in the diagnosis of HCC than AFP. Percentages of AFP-L3 are measured with high sensitivity and hence known as the most important early predictor at low AFP levels. One of the significant biomarkers is DCP (Des-gamma-carboxy-prothrombin) that is relatively more accurate than the other two diagnostic pointers. While differentiating malignant HCC with nonmalignant liver disease, it was clear that the diagnostic value of DCP is better than AFP. DCP is a great deal more effective than AFP in diagnosing HCC on the comparatively lower level. When compared these diagnostic carcinoma biomarkers, showed the potency in a descending order from DCP>AFP> AFP-L3.


Introduction
Hepatocellular carcinoma (HCC) elucidates for more than 85-90% of primary cancer of liver and causes many deaths worldwide [1]. Among the type of cancers, it is at number six in the world [2]. It kills annually more than 650,000 people in the world [3]. HCC was known as the second most lethal cancer [4].
In early stages liver cancer shows no symptoms, after its adequate progression clinical symptoms are noticeable. Earlier studies predict that analysis of HCC at the initial stage and its efficient treatment enhance the lifetime of patients having liver cancer. 60-80% patients of HCC have a condition called cirrhosis [5]. Patients of HCC which have liver cirrhosis, lead to chronic liver disease. In the case of liver cirrhosis there is a decrease in proliferation of hepatocytes that reduce the liver regenerative capacity and leads to fibrous tissues and damage to liver cells which then causes cancerous nodules [6].
Myofibroblasts are the unique population of smooth muscle-like fibroblasts. These cells play a role in the secretion of growth factors, wound healing, matrix deposition and degradation. That's why myofibroblasts help in both human physiology and pathology. Fibroblasts of cancer tissues are similar to myofibroblasts [7]. In the wound healing process cancer-associated fibroblasts (CAFs) are active just like normal fibroblasts and thus promotes tumor growth by secreting cytokines and growth factors [8]. Chronic HBV and HCV cause hepatic fibrosis and results in the development of HCC. Worldwide, HCC is mainly caused by chronic hepatitis B while its primary cause is hepatitis C. Individuals that are suffering from hepatitis B can develop HCC without any cause of cirrhosis [9]. For HCC finding, various types of innovative techniques like imaging apparatuses are accessible for clinical staff to utilize. These advances involve Digital or computerized tomography and resonance imaging and use of tumor biomarkers, similar to alphafetoprotein (AFP), Golgi protein 73, and Vit-K absence or antagonistic-II. As of now in clinical practice, serum AFP levels are for the most part utilized by numerous specialists for diagnosing HCC. Even though that, serum AFP levels are additionally higher in generous liver diseases, similar to liver cirrhosis and hepatitis, and in up to 40% of patients with HCC levels of AFP can be typical. That is the reason, the poor explicitness of AFP is a worry for its application in the finding of disease. [10-12].
To cure HCC, surgical resection and liver transplantation is the most efficient way but, in most cases, surgery is not adapted because of intrahepatic and distant metastasis during diagnosis and these are suitable only for small tumors [13].

HCC-diagnostic biomarkers Biomarkers in serum Alpha-fetoprotein (AFP)
The fetal liver produces a specific glycoprotein known as alpha-fetoprotein. After birth concentration of serum falls rapidly and in adulthood its synthesis is repressed. More than 70% of patients of HCC have a higher concentration of AFP in the blood due to the excretion of the tumor. AFP in serum is an important tumor marker in diagnosing HCC patients. The Level of AFP in serum higher than 400ng/ml is measured as diagnostic however such higher percentages are observed in few patients of HCC AFP level in serum, resulting in two main problems; First, gradually increased level of AFP in serum in case of chronic liver disease like hepatitis (>100 ng/ml), chronic hepatitis, acute hepatitis and cirrhosis. Secondly, in up to 40% of patients showed normal levels of these biomarkers mostly during early stages. Thus, it results in false-negative and positive values [18]. AFP in tumor tissues is available in bound state, conjugated to different proteins and furthermore released in the circulatory framework. In vitro and in vivo frameworks created to see the take-up of AFP by tumor cells have demonstrated positive outcomes. Tumors of the muscle tissues show endocytosis of AFP corresponding to the fetal and neonatal muscle tissues. As tumor cells like fetal endodermal, ectodermal and mesodermal cells are yet not separated, so it is presumed that AFP take-up happens just in undifferentiated cells and neoplastic cells. Followed by take-up, its essence has been pointed by radio-imaging in cell film invaginations and golgi bodies arrange that encompasses the core [19]. Lens Culinaris agglutinin-(LCA) According to bind capacity to lens Culinaris agglutinin (LCA), there are three distinct sorts of markers that change just in sugar chains (AFP-L1, AFP-L2, AFP-L3). In serum of non-threatening incessant liver illness patients, the significant sort of AFP is AFPL1 (not bound to LCA part). Rather than that, the most important type of AFP in HCC patient's serum is Lens Culinaris-reactive AFP, also called AFP-L3. AFP-L3 is a fucosylated specie of AFP (an isoform of AFP) which is the product of GDP-fucose in the presence of alpha 1-6 fucosyltransferase. In HCC tissues, the activity of this enzyme is higher than normal tissues, so AFP-L3 is known as more specific in the diagnosis of HCC than AFP. An increase in AFP-L3 levels indicates HCC in patients along with the constant increase in serum AFP level [20]. With the help of a newly developed immunoassay system, AFP-L3 percentages are measured with high sensitivity. This high sensitivity of AFP-L3 is used as a predictor in the early stages of HCC observation. High level of AFP-L3 is considered as an early prognostic factor in HCC development even at low serum concentrations of AFP as well as in the absence of ultrasound findings [21]. Lens culinaris agglutinin-reactive AFP (AFP-L3) is a glycoform variation of AFP and is communicated as a level of the all-out AFP level. It tends to be identified in the serum of roughly 33% of patients with little HCCs (< 3 cm) where sliced off degrees of 10% to 15% are utilized. At higher cut-off degrees of > 15%, AFP-L3 shows an affectability of 75% to 96.9% and explicitness of 90% to 92% [60,61]. The utility of this marker is restricted as studies have just been led in East Asian populaces in whom AFP levels are as of now raised [22]. Des-gamma-carboxy-prothrombin (DCP) or (PIVKA-II) DCP is a protein present in serum that is secreted by HCC cells thus commonly used in the prognosis of HCC. It is known as, abnormal prothrombin induced by the absenteeism of a fat-soluble vitamin that is K or antagonist II (PIVKA-II) that lack 10glutamic acid carboxylation on its Nterminal. DCP thus shows a lack of coagulation activity.
As it lacks carboxylation of carbon at its γ-position thus known as des-γ-carboxy prothrombin. DCP activates the proliferation of HCC cells [23]. The malignant potential of DCP has been described as an autologous growth factor that triggers HCC growth and thus during hepatocytes malignant transformation, it activates and increases angiogenic factors like endothelial growth factor receptor and vascular endothelial growth factor (VEGF). DCP activates HCC progression through stimulation of the DCP-Met-JAK1-STAT3 signaling pathway. Metastasis and HCC invasion are increased by activation of matrix metalloproteinase (MMPs) signaling pathway [24]. In patients of HCC, increased levels of DCP are found. DCP has high sensitivity and specificity in the detection of HCC [25]. Des-gamma-carboxy prothrombin (DCP), otherwise called protein actuated by nutrient K nonattendance II (PIVKA-II), has been researched solely in Asian nations as a helpful liver tumor marker over the most recent 2 decades. DCP contrasts from prothrombin by the arrangement of aminocorrosive deposits, coming up short on the capacity to associate with other coagulation factors. This "irregular prothrombin" was at first found in the blood of patients introducing nutrient K insufficiency or accepting nutrient K enemies. In 1984, Liebman et al detailed just because that serum DCP was likewise expanded in HCC patients. The specific component by which DCP is created by the tumor isn't yet completely saw, yet the standardization of DCP levels after healing malignant growth treatment obviously shows that the tumor is the wellspring of its creation [26].

Gamma-glutamyl transferase (GGT)
Gamma-glutamyl transferase (GGT) is an enzyme that bounds to membrane and involved in nucleic acid metabolism, biotransformation and tumorigenesis. High level of γ-GGT levels in serum results in increased cancer risk [27]. It helps in the breakdown of glutathione (GSH) extracellularly. Many tissues produce this enzyme but in serum higher level of GGT is derived from the liver. GGT is carried out by albumin and lipoproteins in the serum [28]. Increased activity of γ-glutamyl transferase (GGT) results in liver injury and mortality in the general population. Although it is present in many tissues only the GGT of the liver is detectable in the serum [29]. Its level increments in the event of liver ailment. It is extremely fundamental for the endurance of people and assumes products jobs in elements of epithelial cells for example liver and kidney. The degree of GP73 in the serum of HCC patients is higher and, in this way, it is otherwise called a serum marker for the location of HCC [30].

Golgi proteins 73 (GP73)
The epithelial cells of numerous human tissues regularly express a protein called Golgi protein-73 (GP73) that is a sort II Golgi-confined vital film protein [31]. Its level increments in the event of liver ailment. It is extremely fundamental for the endurance of people and assumes products jobs in elements of epithelial cells for example liver and kidney. The degree of GP73 in the serum of HCC patients are higher and, in this way, it is otherwise called a serum marker for the location of HCC [32]. The strategies for looking at the GP73 incorporate Western blotting, immunohistochemical staining and enzyme linked immunosorbent assay (ELISA). a few specialists utilized Western blotting to identify GP73 in the serum of sound individuals and liver malignant growth patients, and they found that the affectability was 78% and particularity was 93.5% when hatched with polyclonal antibodies. The affectability when brooded with monoclonal antibodies was 84.7%, and explicitness of 93.5%. The solidness and reproducibility of monoclonal antibodies are better than that of polyclonal antibodies.
[33]. The diagnostic serum levels of biomarkers are shown in the Table 1. In patients of HCC, GPC3 is overexpressed both at quality and protein levels, and its demeanor conjecture a poor prognosis. Earlier examinations indicated that GPC3 capacities in HCC intrusion by the official to atoms like Wnt flagging proteins and development factors. Moreover, GPC3 has been utilized as an objective for atomic imaging and remedial mediation in HCC. As of not long ago, GPC3-focused on attractive reverberation imaging, near infrared imaging and positron emission tomography have been explored for the discovery of HCC [37]. Until this point in time, GPC3-GPC3targeted magnetic resonance imaging, positron emission tomography, and nearinfrared imaging have been explored for early HCC location, and different immunotherapeutic protocols focusing on GPC3 have been created, including the utilization of acculturated anti-GPC3 cytotoxic antibodies, treatment with peptide/DNA immunizations, immunotoxin treatments, and hereditary treatments [38]. Heat shock proteins 70 (HSPs) Heat shock proteins are found ubiquitously within the cells and they act as an influential molecular chaperone in case of stress like carcinogenesis. These are known as extremely conserved proteins, which under normal conditions are expressed at low levels but mostly activated in response to cellular stresses. HSPs play a vital role in apoptosis, protein homeostasis, invasion and signals transduction at the cellular level. In heat shock response, stimulation of HSPs leads cancer cells to adapt to stress conditions and thus due to continuous stress environment in tumor cells, many human cancers show overexpression of HSPs [38].
HSPs are overexpressed in cancer cells that are necessary for their subsistence in lethal conditions. The role of HSPs-70 in HCC is poorly understood although higher intracellular concentration of HSPs results in HCC, thus considered as a useful marker for the progression of HCC. It has been exemplified that higher level of HSPs may act as diagnostic and prognostic markers in HCC [43, 44]. HSP47 is encoded by the SERPINH1 gene, which is situated on chromosome 11q13.5, one of the most every now and again intensified areas in human malignant growth. The adjusted articulation levels of the HSP47 have been related with a few kinds of malignancy, for example, liver, cervical, bosom, pancreatic and gastric tumors. Studies have indicated that the HSP47 advances tumor angiogenesis, development, movement and metastatic limit [45].

Tumor associated glycoproteins 72 (TAG-72)
The tumor-related glycoprotein (TAG-72) is a layer glycoprotein complex of roughly 220-400 kDa that is available on the outside of numerous malignancy cells [46]. It is indicated that TAG-72 is overexpressed in different kinds of human tumors, for example, colon disease, ductal bosom malignant growth, lung malignant growth, epithelial ovarian malignancy, hepatocellular carcinoma, and oral malignancy, however isn't communicated by most typical tissues [47].

Ki-67 antigen
In proliferating cells, a nuclear antigen is present that is known as Ki-67. In cancerous cells, it is one of the most widely used proliferation-associated markers. Various studies suggest that Ki-67 had a close connection with metastasis. Researchers found Ki-67 score was an independent prognostic molecular marker to predict distant metastasis in soft tissue sarcoma [48].

The higher the expression of Ki-67 indicates the faster proliferation of tumor cells [49].
Different tumor biomarkers along with their sensitivity, specificity, and serum level is given in the Table 2. Chronic liver cirrhosis sponsors to HCC because in this case hepatocytes growth and proliferation are waned or decreased that results in tumor formation. The aim of this appraisal is to discover the biological tools for the prevention of HCC. In tissues and body fluid of the human body, there are biomolecules and in case of abnormal conditions like carcinogenesis the level of these biomolecules is altered. So, these molecules are called biomarkers and are known as prognostic tools in the early diagnosis of HCC. Many types of biomarkers have been detected in various stages of HCC that includes enzymes, glycoproteins, peptides, mRNAs and miRNAs that are either obtained from serum or liver tissues of HCC patients. Past assessments showed that DCP is clearly preferable tumor marker over AFP and AFP-L3. The outcome of AFP and AFP-L3 are comparative. Regardless, there is no need for AFP-L3 in the being of AFP. By relating estimations of DCP, AFP and AFP-L3 in the examination of HCC, the result was orchestrated by DCP from the start then AFP and AFP-L3 at third. AFP-L3 is basic in the early affirmation of tumors. DCP had a quick association with tumor size and its critical levels are not found in any patients without HCC. That is the reason DCP is increasingly sensitive and has positive prescient worth (PPV) in the determination of HCC. In this manner, for HCC identification, DCP is utilized as principal serum test. In the serum of HCC patients, both AFP and DCP are performing self-rulingly and contend with each other for the discovery of HCC. The mixes of AFP and DCP help in aggregate affectability and explicitness hence commonly utilized for HCC analysis. If there should be an occurrence of early acknowledgment of HCC, AFP is viewed as more predominant than DCP. Be that as it may, if there should be an occurrence of attacks, DCP is more trustful than AFP as pinpointing or demonstrative device for HCC.