Circulating tumor DNA and its role in detection, prognosis and therapeutics of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is considered the fifth most prevalent cancer among all types of cancers and has the third most morbidity value. It has the most frequent duplication time and a high recurrence rate. Recently, the most unique technique used is liquid biopsies, which carry many markers; the most prominent is circulating tumor DNA (ctDNA). Varied methods are used to investigate ctDNA, including various forms of polymerase chain reaction (PCR) [emulsion PCR (ePCR), digital PCR (dPCR), and bead, emulsion, amplification, magnetic (BEAMing) PCR]. Hence ctDNA is being recognized as a potential biomarker that permits early cancer detection, treatment monitoring, and predictive data on tumor burden are subjective to therapy or surgery. Numerous ctDNA biomarkers have been investigated based on their alterations such as 1) single nucleotide variations (either insertion or deletion of a nucleotide) markers including TP53, KRAS, and CCND1; 2) copy number variations which include markers such as CDK6, EFGR, MYC and BRAF; 3) DNA methylation (RASSF1A, SEPT9, KMT2C and CCNA2); 4) homozygous mutation includes ctDNA markers as CDKN2A, AXIN1; and 5) gain or loss of function of the genes, particularly for HCC. Various researchers have conducted many studies and gotten fruitful results. Still, there are some drawbacks to ctDNA namely low quantity, fragment heterogeneity, less stability, limited mutant copies and standards, and differential sensitivity. However, plenty of investigations demonstrate ctDNA’s significance as a polyvalent biomarker for cancer and can be viewed as a future diagnostic, prognostic and therapeutic agent. This article overviews many conditions in genetic changes linked to the onset and development of HCC, such as dysregulated signaling pathways, somatic mutations, single-nucleotide polymorphisms, and genomic instability. Additionally, efforts are also made to develop treatments for HCC that are molecularly targeted and to unravel some of the genetic pathways that facilitate its early identification.

Introduction

Hepatocellular carcinoma (HCC) or hepatocarcinoma or hepatoma constitutes 90% of all primary liver cancers. It is burningly the third most common disease that results in mortality and the fifth most familiar cancer globally. Low survival rates in HCC are primarily a result of high heterogeneity in treatment response within each Barcelona Clinic Liver Cancer (BCLC) stage of the illness with a shortage of prognostic factors to optimize treatment allocation. Liver cirrhosis ultimately causes HCC and is marked in 85% of liver cancer patients. It could manifest as a diffusely infiltrative tumor that causes vascular invasion, multifocal tumor, or unifocal tumor, which leads to stimulation of angiogenesis. It takes about 200 d to duplicate, which declines with the increase in disease progression (1). At diagnostic time, the tumor has reached the stage of intra- or extra-hepatic metastasis, on stage C according to BCLC categorization. The simultaneous growth of multiple distinct tumors or intrahepatic metastasis and the formation of satellite tumor nodules by the migration of cells that are malignant from an individual parent tumor, may be the cause of multiple nodules occurring concurrently in HCC. The two potential processes of multiple HCC development indicate significant pathophysiology differences that may affect prognosis and treatment (2). There are likely differences in prognosis between multiple HCCs that arise from intrahepatic metastasis and multiple HCCs composed of numerous independent tumors that originate more or less concurrently. The latter kind of multiple HCCs is more aggressive and poorly differentiated. The consequential peril for HCC includes alcoholic liver disease, viral hepatitis (hepatitis B and C), non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD) (3). Patients with these risk factors should have regular screening for HCC, which would increase the survival of the affected patients. Clinically, the malignancy marker for HCC is called alpha-fetoprotein (AFP). However, its sensitivity (39%−64%) and specificity (76%−91%) have substantial limits. Serious signs of HCC include AFP levels higher than 20 ng/mL and lower but steadily rising levels. If positive on imaging, values above 200 ng/mL are very suspicious for the presence of HCC. Des-gamma-carboxyprothrombin, glypican-3, and AFP fractions are a few potential markers that have been researched but are not yet employed in clinical practice (4-6). Viewpoints are no more constrained in detecting protein levels to optimize evaluations for HCC; instead, they can concentrate on minute components produced by tumors throughout the carcinogenic process of HCC. The finding established the concept of liquid biopsy, where in specific constituents, like extracellular vesicles (EVs), circulating tumor cells (CTC), circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), and circulating tumor non-coding RNA (ct-ncRNA), and they diffuse into the blood or additional bodily fluids, including the cerebrospinal fluid, urine, sputum, pleural fluid, and ascites. Various methodologies may be employed to separate cells, proteins, chemicals, nucleic acids, and cysts from bodily fluids to gather as much information as they can to help with early diagnosis and track the emergence of diseases (7,8).

Millions of cancer patients die yearly because of the scarcity of diagnostic procedures which poses a challenge to researchers to peep into the detection criterion to aid in diagnosing and monitoring cancer patients. Some of the available techniques have limitations and cause harm to the patients. Hence there is a dire need for accurate diagnoses of the patients. Nowadays, the liquid biopsy technique is emerging and paving the way to determine the pathology of the disease. Cancer diagnosis depends on various pathological examinations such as imaging studies, tissue biopsies, tumor markers, and blood examinations. Among all these, tissue biopsies are the golden technique, however, it is invasive and requires expertise. Studies have illuminated that CTC, miRNAs, and tumor-relevant protein molecules are appropriate tumor biomarkers for cancer liquid biopsy technique (9-12). ctDNA is emancipated by tumor cells in the blood of patients carrying mutations in the original genome of the tumor. Being a noninvasive technique in nature, ctDNA detection in tumor lesions is found to be more sensitive and specific. Moreover, its detection in the blood helps in prognosis and diagnosis of the tumor and aids in targeted therapy (13).

ctDNA is considered as a single-stranded or double-stranded DNA and is thought to exist in both plasma and serum. It acquires cancer-associated characteristics such as DNA methylation, some viral sequences, and single-nucleotide mutations, possibly because they originate from the tumor tissues. Recently a study found that the blood of liver cancer patient possesses both long and short fragment DNA, but the short fragment is associated with tumor aberration (14). Similarly, in breast cancer patients these short fragments are also found (15). Hence, the detection of ctDNA in plasma not only helps in the prognosis of cancer pathogenesis but also aids in targeted therapy because of the feature of characteristic mutations it possesses for the particular primary tumor. Therefore, researchers have gained an advantage in designing new cancer management methods. It is widely accepted as an intriguing biomarker for liquid biopsy cancer prediction and diagnosis. Other markers previously used are prostate-specific antigen (PSA), AFP, carcinoembryonic antigen (CEA), and cancer antigen 15-3 (CA15-3) but their proportion in cancer patients is around 50%−70% as they are already present in the serum of the patients although in small quantities (16-18). The half-life of ctDNA is under two hours whereas the other protein markers can stay in the plasma for particular days. This reflects the more accurate tumor burden of the patients receiving tumor therapy and the recurrence of the disease. Various studies suggest that the level of ctDNA varies at different stages of cancer and co-relates to cancer relapse (13).

Detection of ctDNA

ctDNA could be detected by using two approaches; the first is targeted and the second one is non-targeted. The targeted approach is utilized to detect the already diagnosed genetic alterations as driver mutations that most customarily manifest it in tumor patients and for which there is now tailored therapy available. In non-targeted therapy, no known previous knowledge of mutation is required. To detect this ctDNA, the polymerase chain reaction (PCR) technique is most frequently used and is thought to be the backbone of finding mutations in targeted approaches for which various methods such as digital PCR (dPCR), emulsion PCR (ePCR), bead, emulsion, amplification, magnetic (BEAMing) PCR are utilized (19) as presented in Figure 1 (20). Additionally, next-generation sequencing (NGS) has been employed to examine and compare pre-determined sequences and the de novo synthesis of the sequence assembly (21) (Table 1).

Figure 1.

Liquid biopsy utilizing plasma or blood samples. (A) CTC, free nucleic acids and EV are taken up by bloodstream peripheral to tumors. It is feasible to examine point mutations, amplifications, deletions, epigenetic changes, and translocations in ctDNA by drawing blood samples from cancer patients; (B) The most common techniques for analyzing ctDNA in blood samples include digital PCR, real-time PCR, BEAMing, COLD-PCR and next-generation sequencing (Reproduced with permission from Ref. 20, Elsevier). CTC, circulating tumor cells; EV, extracellular vesicle; ctDNA, circulating tumor DNA; cfDNA, cell-free DNA; PCR, polymerase chain reaction; BEAMing, bead, emulsion, amplification, magnetic; COLD-PCR, co-amplification at lower denaturation temperature-polymerase chain reaction.

Table 1. Techniques used to identify ctDNA and their advantages and limitations.

Techniques used to identify ctDNA	Description	Sensitivity	Advantages	Limitations	Ref.
ctDNA, circulating tumor DNA; WGS, whole genome sequencing; WES, whole-exome sequencing; PCR, polymerase chain reaction; ddPCR, droplet digital PCR; NGS, next-generation sequencing; CAPP-Seq, cancer personalized profiling by deep sequencing; TS, target-panel sequencing; SNV, single nucleotide variation.
WGS	Target whole genome	Moderate to low	Detects innovative mutations, has multiplex capabilities and high throughput diagnosis	Costly and requires bioinformatics analysis support	(22,23)
WES	Target whole genome	Moderate to low	Detects innovative mutations, has multiplex capabilities and high throughput diagnosis	Costly and requires bioinformatics analysis support	(22,23)
Digital PCR	Known and specific regions	Relatively high	Cheaper comparatively	Low throughput and does not detect innovative targets	(22,23)
ddPCR	Known and specific regions	High	Rapid and sensitive	Low throughput and does not detect innovative targets	(22,23)
Allele-specific PCR	Amplify rare mutant DNA sequences	Lower	Easy to use, budget friendly	detect only small number of genomic sequences in sample	(23)
NGS-amplicon	Deep sequencing of entire genome	High (some methods)	Less expensive than other NGS methods	Less extensive, cannot detect rearrangements without customizing assay	(23)
CAPP-Seq	Target only hybrid capture	High	Detects rearrangements and highly sensitive for SNV	Less extensive than WGS or WES	(23)
iDES enhanced CAPP-Seq	Target hybrid capture and integrated error suppression	High	Has flexibility and coverage, highly reliable to detect all aberrations in a single assay	Less extensive than WGS or WES	(23)
TS	Panel-size targeting	Relatively high	Detects innovative mutations, has multiplex capabilities and high throughput diagnosis	Costly and requires bioinformatics analysis support	(22,23)

As ctDNA can be derived from both healthy and tumor tissues, isolating ctDNA from tumors is still challenging because only 0.01% of ctDNA is from the tumor tissue. This limitation can now be overcome by the “ultra-sensitive” assays that can differentiate between the ctDNA and cfDNA. An example of the ultra-sensitive assay is the cancer personalized profiling by deep sequencing (CAPP-seq) which utilizes a capture-based ctDNA method to detect abundant mutations hence boosting the test’s sensitivity about NGS and helping in the detection of the intratumor heterogeneity (24).

Biology of ctDNA

The fundamental diagnostic hallmark is ctDNA which is conditional on the patient’s chromosomal and epigenetic makeup. It is composed of fragments of DNA of 166 base pairs in length and is separated at low concentrations from tumor cells. It is protected from blood-borne nucleases by being wrapped with a single nucleosome. This wrapping with nucleosome enables it to be detected by deep sequencing and mapping of genes and can locate the origin of the ctDNA tissue (25).

Biogenesis of ctDNA

Senescence, active secretion in EVs, mtDNA excretion, and cell death by apoptosis, ferroptosis, pyroptosis, necrosis, oncosis, and phagocytosis represent a few of the processes known to yield ctDNA (26). Passive and active mechanisms have been determined so far to explain the release of ctDNA. According to the passive process, necrotic cells’ nucleic acids are ingested by macrophages and then released into the circulation. Tumoral nucleic acids generated via live cells may be by the active processes (27) shown in Figure 2. cfDNA integrity appears to be higher in cancer patients when compared to healthy individuals, even though ctDNA generated by apoptotic bodies is more precise in terms of tumor molecular information. This implies that necrotic cell death plays a significant role in ctDNA release, especially in advanced stages and aggressive tumors (28,29).

Figure 2.

Biology of ctDNA. ctDNA, circulating tumor DNA.

Clearance of ctDNA

The precise quantity of cfDNA, and specifically ctDNA, is determined by a harmony between DNA leaking and DNA clearance. The three main mechanisms that affect the half-life of ctDNA are: 1) the action of bloodstream DNases; 2) the dynamic removal of DNA and nucleosomes; and 3) organ filtration like the kidney or lymph nodes (Figure 2). The half-life of ctDNA varies between 16 min and 2.5 h. Diverse filtering organs may be employed to carry out ctDNA clearance. The majority of cfDNA is removed by the liver’s kupffer cells, which selectively remove lengthier fragments. The kidneys thereafter engage in DNA fragmentation employing their deoxyribonuclease activity. An insignificant part of ctDNA clearance is also performed by the lymph nodes and spleen’s macrophages. Apart from these organs, lymphatic drainage might serve as the primary means of ctDNA elimination in the tumor microenvironment (28,29).

ctDNA as a potential tumor biomarker

ctDNA is now considered as a potential biomarker and its quantity is markedly raised in patients compared to healthy individuals. Some studies indicate that its value is raised in colorectal, breast, ovarian, lung, and liver cancer and is linked to tumor volume, which lowers overall survival. ctDNA is a particular biomarker that permits early cancer detection, treatment monitoring, and prognostic data on tumor burden subsequent to therapy or surgery. A significant benefit of ctDNA detection over needle biopsies is the capacity to obtain ctDNA noninvasively, facilitating continual patient surveillance (30). Furthermore, ctDNA is utilized for detecting tumor mutations. For instance, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) variations can be noticed in breast cancer cases (31). Also, in lung cancer, genes such as endothelial growth factor receptor (EFGR) and Kirsten rat sarcoma (KRAS) viral oncogene homolog, and numerous variations of ovarian cancer genes can be identified with 97% accuracy (22).

Advantages and limitations of ctDNA

Advantages of ctDNA include: 1) showcasing a thorough analysis of genomic spectrum in response to various tumor areas; 2) advancements in technology rendered analytical assays more sensitive; 3) short half-life of ctDNA facilitates cancer surveillance in real time; 4) more accurate in terms of clinical implications, i.e. more actionable mutations can be detected; 5) maintain tumor specific genetic mutations hence providing individualized snapshot of disease; and 6) it’s noninvasive. Limitations are as follows: 1) burden on pocket and time-consuming; 2) the majority of recently developed assays are still awaiting clinical validation; 3) just genetic information, not details about the cancer’s physical location; 4) not appropriate for functional assay; 5) noises from normal ctDNA; 6) difficulties in standardizing methods; and 7) expression of proteins and co-localization are non-existent.

Molecular aspects of ctDNA in HCC

Variables that contribute in genesis, development, and metastasis of HCC have been proposed to develop genetic and epigenetic anomalies. You can better comprehend hepatocarcinogenesis and streamline the arbitrary research of the pathogenic process that underlies HCC providing a synopsis of the molecular terrain related to HCC in ctDNA. The insertion of hepatitis B virus (HBV) and favored end motifs or coordinates shows that signal copy number variations (CNVs), single-nucleotide variations (SNVs), and DNA methylation aberrations are the main molecular changes in the ctDNA of HCC (Figure 3) (32). Cancer genome sequencing, which commenced in 2006, has generated trustworthy and thorough tumor genome data, enabling an optimal setting for the expansion of ctDNA testing. Globally, an enormous effort and monetary investment are being made to use this “liquid biopsy” in clinics in conjunction with the advancement of sequencing technology. In HCC patients, ctDNA testing may be employed for a variety of purposes, such as 1) early cancer detection; 2) evaluating the spread and heterogeneity of the tumor; 3) discovering therapeutic targets; 4) continuous monitoring of disease recurrence and treatment response; and 5) monitoring the surfacing of drug resistance in real time. For these applications, it is crucial to locate epigenetic and genetic mutations in ctDNA that are distinctively linked to various phases of HCC (such as hepatocellular dysplasia, early HCC, advanced HCC, and metastatic HCC) and cause variations in multiple genes. However, owing to substantial technological challenges in ctDNA identification and analysis, particularly in HCC, assessment of the clinical significance of ctDNA in the detection of cancer, medication, and prognosis has been hindered (33).

Figure 3.

A brief description of ctDNA’s molecular milieu and its value for HCC treatment. The same mutations present in tumor cells are also present in ctDNA, which is derived from the tumor tissue. Because ctDNA and tumor cells are consistent, using ctDNA as a liquid biopsy for several aspects of HCC clinical care may be possible (Reproduced with permission from Ref. 32, Elsevier). ctDNA, circulating tumor DNA; HCC, hepatocellular carcinoma.

Various biomarkers of ctDNA in HCC

Several biomarkers have been dug out and studied for their alterations in all types of cancers, including HCC. They are categorized based on aberrations in genetic machinery and their importance in diagnosis, prognosis, and therapeutic implications. The alterations may be SNV (either insertion or deletion of a nucleotide), CNV, DNA methylation, homozygous mutation and gain or loss of function of the gene. The TERT promoter is the site of the prevailing alterations in HCC (60%) and is linked to elevated telomerase production. TP53 and CTNNB1 mutations are the next most common, representing 25%−30% of patients with HCC. Among the genes with low-frequency abnormalities are TSC1/TSC2, RPS6KA3, KEAP1, AXIN1, ARID2, ARID1A and MLL2. Three groups of genes related to risk factors, including TP53 (HBV), AXIN1 and CTNNB1 (alcohol), were discovered investigating the association of mutations (34) (Table 2).

Table 2. Frequently-mutated biomarkers for ctDNA of HCC.

Biomarkers	Alterations	Relevant pathway	Classification	D/P/T	Ref.
ctDNA, circulating tumor DNA; HCC, hepatocellular carcinoma; SNV, single nucleotide variation; CNV, copy number variation; HBV, hepatitis B virus; D, diagnostic; P, prognostic; T, therapeutic.
TP53	SNV (insertion or deletion of bases, e.g., Ser-249)	P53 signaling pathway	Suppressor	D, P & T	(35-37)
TERT	SNV	Telomerase maintenance	Oncogene	P	(35,37,38)
CTNNB1	Mutation	WNT signaling pathway	Oncogene	P & T	(35,39)
AXIN1	Homozygous deletion	WNT signaling pathway	Suppressor	P	(35,40)
CDKN2A	Homozygous deletion/ methylation	Cell cycle	Oncogene	T	(37,41)
ARID1A	Mutation	SWI/SNF complex-related pathway	Suppressor	P	(42)
ARID2	Mutation	SWI/SNF complex-related pathway	Suppressor	P	(34)
RASSF1A	Hypermethylation	MAPK/RAS signaling pathway	Oncogene	D & P	(37,43)
SEPT9	Hypermethylation	Cell division	Suppressor	D	(37,44)
KRAS	SNV	MAPK/RAS signaling pathway	Oncogene	T	(37)
CDK6	CNV (loss of 1p)	Cell division	Not known	T	(37)
EGFR	CNV (loss of 4q)	EFGR signaling pathway	Oncogene	D/T	(37)
MYC	CNV (loss of 8p)	Transcription factor	Proto-oncogene	P/T	(37)
WWP1	Gain of function in cell division	Smad signaling	Oncogene	Poor prognosis	(37)
KMT2C (MLL3)	HBV integration/ hypomethylation	Chromatin remodeling	Suppressor	Early detection of recurrent HCC	(37)
CCNA2	HBV integration/ hypomethylation	Cell cyclin family	Tumorigenic gene	Early detection of recurrent HCC/P	(37)
BRAF	CNV (gain of 8q)	Ras/Raf/MEK/ERK	Proto-oncogene	T	(37)
CCND1	Focal amplification or deletion	P53 signaling pathway	Oncogene	−	(37)

TP53

The most frequent mutation in HCC, the TP53 mutation, influences the course and outcome of HCC. It has been delved into how the TP53 mutation controls the immunophenotype of HCC and impacts the prediction of the disease. The immune response is down-regulated in HCC as a result of the TP53 mutation. In HCC, TP53 mutation is closely associated with the immunological microenvironment (44). Despite sequencing countless genomes of cancer, aspirants of the utmost significance have not been discovered. One of the most obvious changes in common human cancers is TP53 mutation, which is commonly seen. The management of cell cycle and apoptosis after DNA damage are two critical functions of the wild-type TP53 proteins. On the other hand, if the TP53 gene is altered, DNA-damaged cells could evade apoptosis and modify into cancerous cells. Additionally, the mutant TP53 protein gets deprived of its function in the wild-type state and aggregates up in the nucleus. This buildup is thought to be a very accurate indicator of malignant tumors (45). The molecular etiology of HCC is swayed by an assortment of genetic and epigenetic modifications, which include WNT transcription system activation and somatic modifications in the TP53 tumor suppressor gene. Aflatoxin B1 (AFB1) typically triggers C:G to A:T transversions at the third base in TP53 codon 249 and works with HBV to cause p53 alterations in HCC. Both aflatoxin exposure and the likelihood of developing HCC are biomarkers for the possibility of TP53 mutant DNA in plasma. The reactive oxygen/nitrogen species are generated by chronic HBV and hepatitis C virus (HCV) infection, oxyradical diseases including hemochromatosis and other conditions. These species tend to disfigure DNA and modify cancer-related genes (such as TP53). “Gain of oncogenic function” may be observed in specific deviant p53 proteins. A critical defense mechanism against this nitrosative and oxidative stress is the p53 biological network. p53 governs the transcription of defensive antioxidant genes depending on the severity of DNA damage and transactivates the pro-oxidant genes assisting in apoptosis in cases of severe DNA damage (46). TP53 changes are not typically observed in preneoplastic lesions, in comparison to TERT promoter mutations, and their prevalence is increased along the course of premature to progressive HCC (15.5% BCLC-0 vs. 35% BCLC-C) (47).

Telomerase reverse transcriptase (TERT)

Isolated studies have indicated an association between TERT expression abnormalities or promoter mutations in HCC recurrence and cancer progression. HCC is characterized by several alterations, including the TERT promoter mutation, preeminently thoroughly investigated mutations in HCC. Cellular ribonucleoprotein telomerase is an enzyme. It increases telomere length, which stimulates cell proliferation. The telomerase subunit TERT is in charge of the enzyme’s catalytic activity. Expression of this gene is customarily severely constrained in the majority of normal cells, but it is augmented in numerous carcinomas. Both transcriptional-level and posttranslational-level regulation of TERT expression are achievable. Some cancer-associated TERT promoter alterations occur at the transcriptional level via the generation of de novo transcription-factor-binding sites (48). The mutations 124C/T and 146C/T, also known as C228T and C250T, respectively, are two significant TERT promoter mutations. The alleged E-twenty-six (ETS) transcription-factor binding sites produced by these two mutations activate TERT transcription. These changes result in the overexpression of TERT and the restoration of telomerase activity by establishing a binding site for the transcription factors ETS and ternary complex factor (TCF) (49). Approximately more than 60% of HCC patients convey TERT promoter mutations. TERT promoter mutations have been proven to be the prompt recurring somatic genetic variations and the “sentinel” for HCC in the malignant amendment of cirrhotic hepatocytes, which is a multistage process (48). TERT promoter hotspot mutations have a comparable distribution across all BCLC stages (from 50% in BCLC-0 to 60.6% in BCLC-C stage), and they may also be detected in inferior and prime dysplasia nodules (6% and 19%, respectively), emphasizing their involvement in the initial phases of liver carcinogenesis (47).

Catenin beta 1 (CTNNB1)

One of the many key genetic changes in HCC is deregulated Wnt/β-catenin signaling. Comprehensive genomic investigations have shown that roughly 35% of human HCC samples contain gain-of-function mutations of CTNNB1, which encrypt β-catenin, and loss-of-function mutations of AXIN1. Human HCCs with Wnt/β-catenin pathway impetus have distinctive gene expression patterns and clinical characteristics. Activated Wnt/β-catenin engages with several signaling pathways along its downstream effectors to help HCC flourish. Consequently, methods that target Wnt/β-catenin have been scrutinized as potential treatments for HCC (50). The N-terminal domain of β-catenin’s altered phosphorylation sites inhibits the protein’s phosphorylation and subsequent proteasomal breakdown. As a result, β-catenin concentrates in the cytoplasm, inhabits the nucleus, and activates the transcription of Wnt target genes involved in apoptosis and multiplication (51). Early-stage HCC has been analogous with activation of the Wnt/β-catenin pathway and tumor development. There is still debate concerning the relationship between β-catenin activation and patient survival with HCC, and most studies point to the CTNNB1 mutation as a positive prognostic factor. The coding transcript of the gene CTNNB1 includes the variation CTNNB1 rs121913407: c.133T>C (p. Ser45Pro), whereby cytosine is swapped for thymine at nucleotide location 133. This missense mutation results in the proline replacement for serine at amino acid location 45 of the corresponding protein reference sequence. Chromosome 3’s short arm (3p22.1) is where CTNNB1 is found. It is listed in ClinVar as a missense mutation that is pathogenic or possibly pathogenic and a somatic deleterious variant in HCC. Real-time polymerase chain reaction (RT-PCR) was used to evaluate the CTNNB1 gene locus c.133T>C (p. Ser45Pro) mutation. Using 20 ng of isolated DNA and a final volume of 20 µL, RT-PCR tests were conducted by using TaqMan Genotyping PCR Master Mix and SNP Genotyping Assay (51). They utilized PCR reaction mix quantities according to the producer’s recommendations (52). Almost 10%−15% of hepatocellular adenomas show mutations in the CTNNB1 gene’s exon 3, which are linked to an elevated risk of HCC’s malignant progress (47).

Axis inhibition protein 1 (AXIN1)

Initially, it appeared that abnormalities in AXIN1, present in 8%−10% of HCC, supported tumor growth by abnormally increasing β-catenin signaling. AXIN1, a linker protein, was first discovered as a speed-limiting component of the destruction complex and a down-regulator of the officially recognized Wnt pathway. The β-catenin pathway was therefore expected to be abnormally activated in the liver if AXIN1 function was impaired. The genetic program expressed by human HCCs with rendered inactive AXIN1 mutations was later demonstrated to be distinct from that of HCCs with CTNNB1 mutations. Mutations causing AXIN1 loss of function have been assigned to the proliferative class, which was linked with a more belligerent phenotype and a boost of signals of the cell cycle, while mutations causing CTNNB1 were linked to the non-proliferative class, which was associated with a better prognosis (52). A second reason, the AXIN proteins have drawn a lot of attention in the field of cancer research, is that the tankyrase inhibitors may be used as a treatment approach to treat β-catenin-dependent malignancies since they can enhance their activity in the β-catenin destruction complex. Like β-catenin itself, the AXIN proteins are tightly regulated by proteases. As a result of their association with AXIN, the poly-ADP-ribosyl transferases tankyrase-1 and tankyrase-2 (encoded by TNKS/TNKS2) undergo PARsylation, followed by ubiquitylation and degradation, which restricts the activity of the destruction complex. All elements of the β-catenin demolition complex combine by inhibiting tankyrases and forming a so-called degradosome, which promotes an effective β-catenin turnover. The use of tankyrase inhibitors in treating different cancer types has been researched, giving some encouraging first findings for a particular subset of tumors (53).

Cyclin-dependent kinase inhibitor 2A (CDKN2A)

The multi-step nature of HCC and the aggregation of genetic and epigenetic alterations are well known. Additionally, the most frequent epigenetic modification, DNA methylation, has been shown to affect tumor suppressor gene transcriptional silencing and genomic stability in HCC. A widely recognized tumor suppressor gene called CDKN2A produces the CDKN2A protein, through binding to cyclin-dependent kinases (CDKs), and prevents cell cycles from moving from G1 to the S phase. Since Wong’s initial discovery of CDKN2A promoter methylation in the serum and plasma of patients with liver cancer in 1999, more research has been conducted to determine the link between this methylation and the development of HCC (54). CDKN2A encodes the p16 gene and participates in several biological pathways, including those that increase tumor cell growth, decrease tumor cell apoptosis, stimulate tumor stromal angiogenesis, and lessen the sensitivity of cancer cells to chemotherapy. CDKN2A deletions have been identified in 8% of HCCs. A tumor suppressor gene called CDKN2A causes cell cycle arrest in the G1 and G2 phases and inhibits the cancer-promoting effects of MDM2 and CDK4/6. Expression of CDK4 and CDK6 is increased when CDKN2A mutations are prevalent. Independently of other conventional triggers, inactivation of CDKN2A has been associated with a bad prognosis; in addition, CDKN2A changes can be seen in more advanced, aggressive cancers. In advanced HCC, CDK4/6 inhibitors are under investigation due to the frequent loss of CDKN2A (55). Patients with HCC commonly have epigenetic lesions at tumor suppressor and cell cycle regulator genes, such as silencing the CDKN2A locus. As a result of the weakened cell cycle checkpoints in HCC, CDKs (such as CDK4/6) and E2F transcription factors (such as E2F1) may be activated, leading to an unchecked proliferation of HCC cells.

AT-rich interaction domain 1A & AT-rich interaction domain 2 (ARID1A & ARID2)

High levels of mutation are seen in chromosome remodelers in cancer, particularly HCC. These alterations commonly occur in genes that encode for the ATP-dependent SWI/SNF remodelers’ subunits, the AT-rich interactive domain (ARID) genes, bewildering the increasing complexity of the characteristics of the highly versatile BG1/BRM-associated factors (BAF) and polybromo-associated BAF (PBAF) complexes, like ARID1A/B or ARID2. The specific effects of individual gene changes are multiplied by the SWI/SNF complexes’ involvement in various organs and functions, particularly in regulating gene expression. For the most part, frameshift or inactivating mutations in ARID1A are the ones that result in the loss of ARID1A expression. Frame-shifting deletion, nonsense mutation, and splice site modification are the three types of ARID2 mutations prevalent in HCCs associated with HCV (56). ARID1A participates in the cell cycle/DNA damage checkpoint, the modulation of P53 targets, and the turning on of telomerase as an element of tumor suppression. The improbability of HCC patients may be because of the mutation of ARID1A, which in HCC boosts migration and multiplication. Another study showed that ARID1A deletions stimulate the PI3K/mTOR pathway, which boosts HCC cell proliferation, invasion, and migration. Additionally, Li et al. have shown that ARID1A deletion stimulates the development of HCC by increasing MYC transcription (57). The frequency of ARID1A and ARID2 mutations is identical across all BCLC stages, and they are prevalent in HCC associated with alcohol-related liver disease and have been present in up to 10%−15% and 5%−8% respectively of nearly all HCC cases (47).

Ras association domain family 1 isoform A (RASSF1A)

Promoter hypermethylation usually culminates in the silencing of the cancer suppressor RASSF1A in HCC. Misfolded or clustered proteins and damaged organelles undergo elimination by autophagy. To promote cancer, autophagy deficiencies increase oxidative stress and genomic instability. Autophagy flux is triggered by boosting levels of the RASSF1A-interacting microtubule-associated protein 1 S (MAP1S), thereby suppressing HCC and lengthening lifespans (58). The RASSF1A gene, discovered on human chromosome 3p21.3, affects the cell cycle in a number of ways, including by promoting microtubule stabilization, strengthening cellular adhesion and motility, launching apoptosis and proliferation, as well as regulating microtubule dynamics during mitotic progression — all of which aid in the malignant growth of the hepatocyte. In numerous types of human cancer, including HCC, the methylation of CpG sites results in the downregulation of RASSF1A. The presence of methylated RASSF1A results in endogenous transcriptional inactivation and limits its tumor-suppressive capabilities, which may promote the start and progression of HCC on its own (59). RASSF1A’s promoter hypermethylation may be beneficial for screening and is an appealing early diagnostic and prognostic biomarker in HCC. By Mohamed et al. (59), RASSF1A was also found to have a beneficial function in the early detection of HCC, with a sensitivity of 90%. Additionally, studies have found that RASSF1A had a prediction accuracy of 77.5% for separating HCC patients from healthy patients. A significant tumor suppressor with diverse biological activities is the RAS association domain family protein 1A (RASSF1A), whose promoter is frequently inhibited by promoter hypermethylation in a variety of malignant tumors, including HCC. According to some studies, the RASSF1A gene may also regulate microtubules, DNA repair, cell cycle, and apoptosis. As observed in HCC patients, the methylation and inactivation of RASSF1A deploy the most critical cell protective activities inhibiting the Hippo and Wnt signaling pathways (60).

Septin 9 (SEPT9)

A growing body of research has revealed that methylated septin 9 (mSEPT9) DNA, used to diagnose colon and rectal cancers (CRC), could be used to detect HCC. The expression of SEPT9 was discovered to be highly expressed in normal tissue but was usually reduced in HCC by aberrant promoter hypermethylation. It has been discovered that SEPT9 in particular is crucial for developing lipid droplets in the liver. These droplets interact with other cell organelles intracellularly and play a remarkable pathogenic role in the advancement of viral-related cirrhosis and non-alcoholic steatohepatitis (NASH). Preserving cellular activity and organelle signaling pathways is another function of SEPT9. Additionally, mSEPT9 led to the loss of apoptotic cellular activity and was amenable for the activation of hepatic stellate cells, leading to liver fibrogenesis. These are two critical steps in the process of developing hepatocarcinogenesis (61). As an essential epidriver gene in liver carcinogenesis, SEPT9 expression is usually repressed by aberrant promoter hypermethylation in liver cancer. According to recent reports, the mSEPT9 test among cirrhotic patients is an auspicious circulating epigenetic biomarker for HCC detection at the level of the individual patient. mSEPT9 examination for HCC was established by Kotoh et al. with 63.2% sensitivity and 90.0% specificity (62). With the advancement of HCC, the positive rate of mSEPT9 arose. In 2016, the US Food and Drug Administration (FDA) approved Epi proColon (Epigenomics AG, Berlin, Germany), the first commercial blood-based test for colorectal cancer screening based on methylation in DNA testing of SEPT9. Epi proColon does have few drawbacks. To resolve these issues, a previously created co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) assay was applied, which avoids the necessity for bisulfite treatment and methylation DNA immunoprecipitation. Because it is 100 times more delicate than the conventional bisulfide-based methylation assay, this test addresses the issue of inadequate input DNA. The COLD test quantitatively finds even one copy of a methylated gene in a minuscule DNA sample without the need for DNA bisulfite treatment. The most distinctive aspect of this innovative assay is that it combines droplet digital PCR (ddPCR) with three methylation-sensitive restriction enzymes. Patients with colorectal neoplasia, including cancer, described how well the serum CORD test for mSEPT9 was performed. In this investigation, they assessed the assay’s potential use in detecting mSEPT9 in HCC patients (62). In contrast to patients with preliminary-stage HCC (stages A/B), patients with late-stage HCC (stages C/D) had considerably lower plasma mSEPT9 CT values, demonstrating a positive correlation between plasma mSEPT9 and HCC malignancy. As compared to HCC participants (specificity 96.0% and sensitivity 76.7%) and at-risk disease patients (specificity 83.2% and sensitivity 76.7%), plasma mSEPT9 demonstrated astounding performance in diagnosing patients with preliminary-stage HCC according to the receiver operating characteristic (ROC) curve. All of these findings show that plasma mSEPT9 is a highly effective biomarker for diagnosing HCC and early-stage HCC detection in clinical settings (63).

Kirsten rat sarcoma viral oncogene homolog (KRAS)

Notably, 50%−100% of HCCs also have high Ras/Mek/Erk pathway activation levels, which is associated with a poor prognosis. Ras is a key oncogene that is frequently hypermutated in several malignancies, including HCC. The activated Ras cascade that mutant Ras triggers downstream targets of Raf/Mek/Erk and/or PI3K/Akt/mTOR, two important carcinogenic signal transduction pathways, are activated in HCC. These pathways control cell proliferation, cycle, and differentiation. Ras’s three most often mutant isoforms are Nras, Hras, and Kras. Therefore, Kras mutants are intriguing candidates for therapeutic intervention (64). Approximately 30% of all human tumors are found to have a Ras mutation, which most frequently affects the KRAS gene. While H-Ras and N-Ras modifications are also seen at low ends, KRAS mutations have been found in 7% of human liver tumors. Ras signaling activity has been consistently seen in human HCC samples, and it has been demonstrated to cause hepatocellular growth and alterations. A significant portion of KRAS genes exist in exon 2, and the most common mutations are found at codons 12, 13, 146 and 154, accounting for more than 80% of all mutations (65). As dysregulated mTOR signaling plays a valuable role in the initiation and advancement of HCC, targeting the mTOR pathway appears to be a viable HCC therapy approach. While they have very little effectiveness opposed to the advanced HCC in clinical trials, preclinical investigations have shown the first generation of mTOR inhibitors, rapamycin derivatives, to be beneficial in inhibiting HCC growth (64).

Cyclin-dependent kinase 6 (CDK6)

HCC is a terminal form of cancer for which there is no cure. The PI3K/AKT signal pathway and CDK4/6 are crucial in carcinogenesis and provide viable therapeutic targets for HCC. Cyclin-dependent kinases (CDKs) are a member of serine/threonine protein kinases, which are indispensible for managing cell cycle, metabolism, neuro-physiological processes, transcription, cell differentiation, and development. Elevated CDK activities in tumor cells promote cell division, genomic instability ( for example, chromosome deletion and DNA mutations), and chromosome instability, all essential for the onset and spread of cancer. Among them, CDK4/6 labors are essential for controlling G1 phase of cell cycle. It is extensively established that many tumors have significantly increased levels of CDK4/6 expression. According to reports, 66.7% of HCC patients had elevated CDK4 and 46% had elevated CDK6 (66). Up to 73% of HCC cases were shown to have the cyclin D-CDK4-RB pathway under aberrant regulation. As a result, targeting CDK4/6 is a viable strategy for treating HCC, and numerous trials have consistently shown that CDK4/6 inhibitors are effective.

Endothelial growth factor receptor (EGFR)

Human cirrhotic liver tissue and HCCs overexpress EGFR. In 68% of cases of human HCC, there is overexpression of EGFR, which is consistent with aggressive tumors, metastasis, and inferior patient viability. It is a prospective therapeutic target. HCC typically exhibits overexpression of EGFR and activation of EGFR is a possible predictor of HCC cells’ initial resistance. EGFR is a receptor tyrosine kinase family. The EGFR dimers into the asymmetric (activator-receiver) kinase dimer when it binds to members of the epidermal growth factor family. Because of this, the receiver subunit’s tyrosine-kinase domain takes on an active conformation, phosphorylating significant tyrosine residues of EGFR at the c-terminal tail. Diverse malignancies have abnormal EGFR activation by EGFR gene amplification, mutations, and/or overexpression, which has been causally linked to the patient’s poor prognosis (67).

Myelocytomatosis viral oncogene homolog (MYC)

HCC typically exhibits elevated receptor tyrosine kinase methylation and the activation of the transcription factor MYC, among other circumstances. Both genes independently induce the onset and spread of liver cancer. In viral and alcohol-related HCC (approximately 70%), the MYC gene is overexpressed and amplified in several human malignancies. One of the initial processes in the genesis of HCC is the augmentation of the MYC locus. Ectopic MYC expression together with other oncogenes starts and promotes HCC. Genetic modeling of HCC has shown that inhibiting MYC causes tumor reversion, indicating that HCC may be clinging on the MYC oncogene. The MYC family member c-Myc is the most often activated oncogene. A transcription factor called c-Myc attaches to the enhancer box sequence CACGTG and controls more than 15% of human genes. These genes are enmeshed in various biological processes, such as cell division, proliferation, cancer, apoptosis, and metabolism. Two of the most common forms of dysregulation in cancer are c-Myc gene overexpression and amplification. The developments of several cancers, including HCC, and c-Myc dysregulation have been linked in several studies (68).

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1)

WWP1 has been found to block tumor growth factor (TGF) signaling by modulating Smad2 and activated receptor degradation. The active type I receptor was made to degrade when WWP1 interacted with Smad7. Furthermore, WWP1 can interact with tumor growth factor inhibitory factor (TGIF) and Smad2 to promote Smad2 degradation. Enhanced WWP1 levels inhibited TGF-mediated growth arrest, while reduced WWP1 levels prevented Smad2’s degradation and increased TGF-induced gene expression. According to research, WWP1 inhibits TGF-signaling via modifying Smad2 and the degradation of active receptors. When WWP1 and Smad7 interacted, the active type I receptor broke down. Additionally, WWP1 may interact with TGIF and Smad2 to foster the degradation of Smad2. Boosted WWP1 levels prevented the degradation of Smad2 and boosted TGF-induced gene expression, whereas decreased WWP1 levels promoted TGF-mediated growth arrest (69). This indicates that WWP1 may play an oncogenic function in human primary HCC and tend both as a prognostic indicator and a possible molecular therapeutic target.

Lysine methyltransferase 2C (KMT2C)/mixed-lineage leukemia 3 (MLL3)

Cancers of the blood and solid tumors are associated with the tumor-suppressor gene KMT2C, also known as MLL3, which is found in a variety of myeloid cells and epithelia. Single nucleotide polymorphisms (SNPs) in the KMT2C gene have also been associated to several cancer types. KMT2C is located on chromosome 7q36 and encodes nuclear proteins (70). The primary transcript of the relatively big gene KMT2C encodes for a protein is 4,911 amino acids in length and covers 59 exons. KMT2C is an ASC-2/NCOA6 complex (ASCOM) component with histone methylation activity and engages in transcriptional co-activation. Although the actions of type 2 lysine methyltransferases and their sequences are highly similar, KMT2C seems to play an extra role in DNA replication and chromosomal preservation. Downregulation of KMT2C significantly alters DNA damage response and repairs gene activity, impairs homologous recombination-mediated double-strand break DNA repair, and causes DNA instability (71).

Cell cyclin A2 & D1 (CCNA2 & CCND1)

In eukaryotes, the ordinance of the mitotic cell cycle is significantly regulated by the cell cycle-related genes CDK1, CDK5, CDC20, CCNA2, CCND1 and CCNB2. Cyclins A2 and E1 control the cell cycle by accelerating the onset and advancement of S phase. A subset of HCC that exhibits cyclin activation by a variety of mechanisms, including enhancer hijacking, recurrent CCNA2 fusions, HBV, and adeno-associated virus type 2 (AAV2) insertions is identified by numerous researchers. Hundreds of tandem duplications and template insertions habitually activate the TERT promoter, giving rise to a unique structural feature linked to cyclin-driven HCC. These rearrangements are compatible with a break-induced replication process since they are highly enriched in early replicated active chromatin areas (72). Some cancers, including pancreatic ductal adenocarcinoma, stomach adenocarcinoma and HCC, have been found to overexpress CCNA2. Additionally, its overexpression suggests individuals with certain cancer types have a bad prognosis. CCNA2 has been discovered as a tumor therapeutic target enmeshed in the processes of cell proliferation. Malignant cell transformation is associated with the dysregulation of CCND1 and CCNE1, and these two proteins are overexpressed in various tumor forms. Due to a lack of reliable diagnosis indicators, patients are frequently diagnosed at advanced stages of HCC growth. The overexpression of CCND1 may promote the growth and development of tumors in the liver, which could lead to hepatocellular carcinogenesis. New research suggests that CCND1 overexpression or poor autophagy could be risk factors for HCC. A mechanistic investigation reveals that activated autophagy results in the phosphorylation (Thr286) and ubiquitination of CCND1 by glycogen synthase kinase-3 beta, followed by the recruitment of specific phagophores by sequestosome1, the formation of autophagosomes, fusion with lysosomes and destruction (73).

v-RAF murine sarcoma viral oncogene homolog B1 (BRAF)

A serine/threonine kinase called BRAF is proto-oncogene that transmits regulatory signals via the Ras/Raf/MEK/ERK cascade. This route mediates the cellular response to growth signals. BRAF somatic mutations offer an alternate method of the MAPK signaling pathway’s abnormal activation, linked to several human malignancies. The well-documented upregulation of this signaling pathway in HCC correlates with the advanced stage. Oncogenic Ras activates the BRAF gene, amongst the RAF isoforms found in humans, causing synergistic consequences in cells countering to growth factor signals.

Diagnostic value of HCC biomarkers

When looking for drug-sensitive gene markers and identifying potential resistance mechanisms in cancer patients, using ctDNA liquid biopsy is beneficial. In a study by Mody et al. (74), ctDNA testing was administered to 35 HCC patients. In patients with HCC, it was discovered that ctDNA can be a very effective non-tissue substitute for genomic profiling. The ability of ctDNA to maintain extensive somatic data about primary HCC and metastases is thought to be its principal benefit. The benefit of ctDNA is its potential for repetition during treatment, whereas the outline of genetic alteration may alter in the course of time. According to Ikeda et al. (75), ctDNA analysis revealed alterations related to HCC carcinogenesis and development, including numerous intricate signaling networks and genes. In patients with HCC, Zhu et al. (76) evaluated the usefulness of ctDNA in anticipating preliminary postoperative tumor reappearance and tracking tumor burden. They looked at 41 patients with a confirmed radiological diagnosis who had liver resections and recovered. The ctDNA genetic alterations were established to be compatible with the mutations seen in the matching HCC tissue. CTNNB1, TP53, BRAF, NRAS, and NFE2L2 are among the genes that have had mutations found in ctDNA tissues and are important in cancer development. To confirm the predictive usefulness of ctDNA, 96 surgically-treated patients with prime HCC were registered in the study (77). Currently, the excised surgical tissues are used to describe the genomic landscape of HCC patients. The repetitive genomic abnormalities are TERT, CDKN2A, CTNNB1, TP53, AXIN1, ARID2, ARID1A, and MLL2. In one study, 27 of 48 pre-operative specimens of patients with early-stage disease exhibited at the minimum of one mutation in TP53, CTNNB1, or TERT, according to an examination of ctDNA mutation detection. TERT promoter (51%), TP53 (32%), CTNNB1 (17%), PTEN (8%), TSC2 (6%), KMT2D (6%), ARID2 (6%), and AXIN1 (6%), as shown by targeted ultra-deep sequencing, were prevailed in ctDNA study of 121 individuals with advanced HCC. In addition to being discovered in mononuclear cells of blood samples or normal HCC tissues, peripheral blood from HCC patients has been reported to harbor at least one of the recurrently mutated loci situated in TP53 c.747G>T (p.R249S), CTNNB1 c.133 T>C (p.S45A), CTNNB1 c.121A>G (p.T41A), and TERT c.-124C>T. Research into the molecular mechanisms fundamental to the aforementioned altered genes in tumorigenesis and development provides broad opportunities for the clinical application of targeted therapy. These studies help screen targeted populations and choose therapeutic medications. Implementing altered genes in early tumor detection is limited since cancer-related sequences discovered by liquid biopsy frequently involve renowned mutations, which have already demonstrated clinical significance. Cohen et al. (78) linked ctDNA mutations with circulating proteins for several prevalent cancer types for early tumor identification. Among 44 liver cancer patients in a cohort, the sensitivity for HCC was about 95%, and it diagnosed nearly all of HCC patients in stage I. First reported by Wong et al. (79), the p15/p16 detection rate can reach 92% and the arithmetic progression of methylation is 48% in p15 and p16. By Mohamed et al. (59), RASSF1A was also found to have a beneficial function in the early detection of HCC, with a sensitivity of 90%. Since no relevant genomic profile has been found to date, analysis of a patient’s mutational genomic landscape may be able to provide personalized treatment. For instance, Ikeda et al. (75) used a commercial NGS panel to assess 14 patients. Researchers found that individuals with advanced HCC who had mutations that trigger the MET and PTEN pathways might gain benefit from sirolimus and cabozantinib, two significant pathway inhibitors. Following the administration of celecoxib (a Cox-2/Wnt inhibitor) and palbociclib (a CDK4/6 inhibitor), AFP levels were reduced in patients with CTNNB1-activating mutations and CDKN2A-inactivating mutations. Additionally, as the intensity of the treatment changes, the gene sequence of the ctDNA might vary. In theoretical terms, the genetic information contained in ctDNA is indistinguishable about the tumor as the primary tumor cells from which it arose. Because of the quick clearance, it serves as a real-time biomarker (80). In a recent study, the ctDNA of a patient receiving capecitabine showed a reduction in the initial BRCA2 and ARID1 mutational alleles after pervasive treatment and the appearance of TP53 anomaly after illness progression (81). Without being found in normal HCC tissues or mononuclear cells of blood samples, at minimum one of the recurrently mutated loci located in TP53 c.747G>T (p.R249S), TERT c.-124C>T, CTNNB1 c.133 T>C (p.S45A), and CTNNB1 c.121A>G (p.T41A), has been found in the peripheral blood of HCC patients (82). The most significant advantage of ctDNA is thought to be its capacity to preserve extensive somatic data concerning the initial HCC and metastasis. The advantageous aspect of ctDNA is that it can be redone during treatment, even though the spectrum of genetic modifications can change over time. The detection of ctDNA in early prognosis of HCC is enhanced by the detection of the biomarkers by liquid biopsy. It was demonstrated that the ctDNA’s genetic alterations matched the mutations found in the corresponding HCC tissues. Researchers identified mutations in the following genes: CTNNB1, TP53, KRAS, BRAF and NFE2L2, which were investigated in ctDNA tissues and have implications in carcinogenesis (83).

Limitations of ctDNA

A few prevalent ctDNA restrictions include the low amount, fragment heterogeneity, low stability, differential sensitivity, limited copies, lack of universal setup, and the likelihood of erroneous results.

Low quantity

Quantity is the main drawback of the current method. Low levels of ctDNAs are excreted by tumor cells. According to studies, the average amount of ctDNA is between 10 and 100 ng, which is comparatively extremely little.

Heterogeneity in fragments

The process of destroying tumor cells results in random pieces, which is the first thing to understand about the current marker, meaning that various samples will yield varying quantities, sizes, and types of pieces. For instance, we may obtain the TP53 and BRCA1 gene segments from one sample but not another. Typically, a fragment’s size falls between a hundred and a few thousand base pairs.

Stability

ctDNA sample has up to 2 h of shorter half-life, making it less stable. As a result, we are forced to process data quickly, which results in inaccurate results and failed assays. The cell’s inherent clearance process kicks into high gear as soon as the ctDNA begins to release. These results refer to blood-derived ctDNA; nevertheless, research shows that ctDNA from other sources, such as plasma, has a relatively greater half-life and can be stabilized for up to 48 h.

Differential sensitivity

Although ctDNA is mainly found in blood, it can also be obtained from other tissues. According to studies, the analytical effectiveness of the test is constrained by the fact that the sensitivity of ctDNA testing differs among sample types. Since blood biopsy is commonly available, simple to isolate, and largely minimally invasive, more research has been done on it. It is more delicate. However, the yield of ctDNA from blood is also incredibly low (84).

Lack of universal setup

There is not a single setup, standard operating procedure, or working method for ctDNA testing, however, there are a lot of known standard operating procedures (SOPs) for isolating ctDNA from blood. It’s also critical to understanding how difficult it is to create a SOP for ctDNA testing.

Methodological limitations

The future of tumor testing may lie in ctDNA. Recent evidence also demonstrates great scope from a clinical standpoint; however, ctDNA testing’s overall sensitivity and accuracy are quite low when compared to other testing approaches.

Limited mutant copies

In this situation, a low copy number is a significant obstacle. Less tumor DNA is lost by cells, and hence there are also fewer copies available for analysis. Even with susceptible digital droplet technology, findings need to be shown by at least three mutant allele molecules.

Limited standards

Since the technique is still developing, no standards, cut-off values, or other testing factors can be used to compare the results (84).

Future prospects

Precision oncology has transformed medicine thanks to the ongoing improvement of molecular tumor knowledge and the regular advancements of molecular technologies. Competitive benefits for precise diagnosis and individualized treatment exist with noninvasive liquid biopsy. By continuously collecting samples, liquid biopsies can give researchers deeper and individualized information about everything from cancer diagnosis to tumor monitoring. However, there are still difficulties in the transfer of liquid biopsy from the bench to the bedside. In the time to come, we could assist patients in avoiding needless medications, or if they are really at high risk, they could get something that provides a significant benefit and changes the course of their treatment. For liquid biopsy to be used clinically, reliable biomarkers and consistent detection techniques are needed. There are various applications for ctDNA in the treatment of cancer. As an excellent prognostic tool, firstly they can assist in determining the likelihood of recurrence (Figure 4). Numerous studies have exhibited that CTCs can be used to assess a patient’s potential relapse risk. Second, liquid biopsies might influence the choice of treatment. Conclusively, liquid biopsy appears to be a practical, noninvasive, and promising treatment for HCC.

Figure 4.

ctDNA biomarkers of HCC in diagnostic, prognostic and therapeutic measurements. ctDNA, circulating tumor DNA; HCC, hepatocellular carcinoma.

Conclusions

The progressive rise in HCC incidence in recent years sparked alarm on a global scale. Additionally, neither the sensitivity nor the specificity of conventional diagnostic liver cancer indicators is preferable, which is unfavorable to the identification and governance of HCC. Therefore, this review sought novel HCC diagnostic, prognostic, and therapeutic targets concerning ctDNA. In this regard, identifying important biomarkers that influence the maturing of chronic liver disease into HCC can bring about the discovery of fresh methods for the targeted elimination of HCC tumors. Therefore, discovering key epigenetic pathways necessary for the growth and maintenance of HCC is an exciting research area for the future development of liver cancer therapies. A blood sample may detect gene alterations that can be treated with particular medications or seek markers that qualify a patient for a specific treatment. DNA fragments from dense tumors permeate the bloodstream through both active and passive mechanisms. It can happen at any stage of the tumor and provides slightly invasive access to important molecular data about the tumor, such as genomic (CNV or point mutations) and epigenetic (DNA methylation alterations) information. Two significant obstacles exist when undertaking mutation identification from the plasma of HCC patients: the genomic complexity of HCC, which is characterized by a broad continum of various putative driver mutations, and the low concentration of tumor DNA among the pool of cfDNA. Several methods can be applied to determine plasma mutations; targeted sequencing enables analysis of a larger panel of potential candidates, whereas ddPCR is better suited to focusing on a small number of genes. Several studies on the TERT promoter found that TP53 and CTNNB1 are hot mutated genes in HCC.

The predictive significance of ctDNA discovery, related to worse survival or increased recurrence, was further verified by targeted sequencing of multiple gene panels. ctDNA from HCC patients has been demonstrated to have altered DNA methylation. For instance, in retrospective research, hypermethylation of RASSF1A, p15, and p16 was recommended as an early diagnostic technique. Numerous studies have advertised that methylation changes in multiple plasma/serum genes, including p15, p16, GSTP1, INK4A, RASSF1A, and others, can identify HCC from controls. The mSEPT9 test for HCC was created by Kotoh et al. (62) and indicates 63.2% of sensitivity and 90.0% of specificity. Generally, the most common somatic events in human cancers are the mutation of genes encoding parts of chromatin remodeling and modifying complexes. For instance, heterozygous deletions encompassing KMT2C occur in a subgroup of aggressive leukemia, while missense and nonsense mutations occur in a variety of solid tumors. In conclusion, it becomes evident to look for such modifications in the ctDNA of HCC for better medication.