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Ding Jingnuo, Zhao Weifeng, Department of Infectious Diseases, Suzhou University First Affiliated Hospital, Suzhou City, Jiangsu Province, 215000 Tel. tumors of the digestive system with a 5-year overall survival of 14.1%. Many patients with HCC are diagnosed at an advanced stage, so early screening is essential to reduce mortality from HCC. In addition to commonly used detection indicators such as serum alpha-fetoprotein (AFP), lens lectin-reactive alpha-fetoprotein (AFP-L3), and abnormal prothrombin (vitamin K deficiency-induced protein II, PIVKA-II), fluid biopsy techniques It has been shown to be of diagnostic value in the detection of HCC. Compared to invasive procedures, fluid biopsy can detect circulating malignant metabolites. Fluid biopsy techniques detect circulating tumor cells, circulating tumor DNA, circulating RNA, and exosomes and are used for early screening, diagnosis, and prognostic evaluation of HCC. This article reviews the molecular biology and application of various fluid biopsy techniques to isolate promising biomarkers that may be viable options for early assessment of HCC to improve early screening of high-risk HCC groups. Keywords: fluid biopsy technique, hepatocellular carcinoma, high-risk group .
Hepatocellular carcinoma (HCC) is a common malignant tumor of the digestive tract, ranking sixth among new cases of malignant tumors in both men and women. 1 Worldwide, liver cancer is the third leading cause of cancer death after lung and colorectal cancer, accounting for 8.3% of cancer-related deaths from all malignant neoplasms. 1 The prognosis of HCC is closely related to the stage at diagnosis. The main reasons for poor survival in HCC are intrahepatic metastases, portal venous tumor thrombi, and distant metastases precluding resection, and many of these characteristics are already present in patients at the time of diagnosis.
Based on diagnostic and treatment guidelines, the main risk factors for developing HCC are cirrhosis of the liver, chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, alcoholic fatty liver disease, and non-alcoholic fatty liver disease (NAFLD). 2 In addition, risk factors for HCC include aflatoxin-contaminated food intake, schistosomiasis, other causes of cirrhosis, a family history of liver cancer, diabetes, obesity, smoking, and drug-induced liver injury. 35- and 45-year-old high-risk groups should have regular medical check-ups. Early screening is an important early treatment strategy to improve the overall survival of patients with HCC.
Biomarkers such as AFP, AFP-L3 and PIVKA-II are recommended for early screening of HCC3,4. Liquid biopsy techniques have shown promising results in early diagnosis and treatment evaluation. 5,6 Significant progress has been made in HCC liquid biopsy, which may have higher sensitivity and specificity than commonly used serum markers such as AFP (Table 1).
AFP is a widely used biomarker in HCC and is currently the most detailed biomarker widely used for early screening, diagnosis, and evaluation of the disease. A persistently elevated AFP level is considered a risk factor for the progression of HCC. 7,8 The detection rate of small hepatocellular carcinoma (sHCC) is increasing with the development of ultrasound and computed tomography, and AFP has been found to be particularly insensitive to the detection of hHCC in clinical practice. According to a retrospective multicentre study9, AFP positive was found in 46% (616/1338) of HCC cases and 23.4% (150/641) of sHCC cases. In addition, AFP levels are elevated in patients with chronic liver disease and cirrhosis. 10 Thus, AFP has a limited screening effect for sHCC. 11 According to the Asia-Pacific Clinical Practice Guidelines for Hepatocellular Carcinoma, the use of AFP is not recommended.12 Clinical evidence suggests that PIVKA-II is superior to AFP in the treatment of HCC and that the combination of PIVKA-II and AFP has a higher diagnostic value in HCC. 13 Compared to tissue biopsy, fluid biopsy primarily detects tumor-associated metabolites in body fluids (blood, saliva, pleural fluid, cerebrospinal fluid, or urine) and is less invasive to tissues. 14 In addition, liquid biopsies may reflect malignant features not present in primary tumor tissue. 15 Liquid biopsies are not yet being tested in clinical practice for all types of tumors, but their diagnostic potential in cancer is attracting the attention of oncologists. 16 Fluid biopsy can detect circulating tumor cells (CTCs), circulating tumor DNA (cDNA), circulating free RNA (ecRNA), and exosomes. In this article, we will discuss the characteristics, role, and application of various fluid biopsy techniques in early screening of high-risk HCC groups.
Extracellular DNA (cfDNA) in blood samples from healthy individuals was first described in 1948 by Mandel et al. 17 cfDNA is a cell-free DNA fragment approximately 160–180 bp in length, originating primarily from lymphocytes and myeloid cells. ctDNA is a specific mutant DNA fragment released by tumor cells into the peripheral blood, which represents the genomic information of tumor cells after certain pathophysiological processes, including necrosis, apoptosis, and excretion. The proportion of ctDNA in total cfDNA varies widely with tumor type, and cDNA fragments are reported to typically be less than 167 bp in length. 18 Underhill’s study showed that cfDNA fragments are generally shorter than normal cfDNA.19 Compared to healthy individuals, the total length of cfDNA fragments in the blood of cancer patients is shorter, so cfDNA can be used as an indicator of early tumor screening. Enrichment of certain subsets of cfDNA fragment lengths can improve the detection of cDNA associated with non-metastatic solid tumors. Studies have shown that ctDNA is found in over 75% of advanced pancreatic, colon, bladder, gastrointestinal, liver, ovarian, breast, melanoma, and head and neck cancers. 20,21 However, the amount of ctDNA in the blood depends on the location of the tumor. 22 In a study by Bettegoud, patients with colorectal, breast, liver, lung, and prostate cancers were found to have higher levels of cDNA in their blood than other cancers. In contrast, in patients with oral cancer, pancreatic cancer, gastric cancer, and glioma, cDNA concentration in the blood was lower. twenty one
Because ctDNA contains the same genetic mutations as primary tumor cells, cDNA can be used to detect heterogeneous tumor-specific mutations and epigenetic changes, including methylation, hydroxymethylation, single nucleotide variations, and copy number variations. twenty three
DNA methylation is one of the most common epigenetic changes resulting in gene repression. Compared with normal cells, there are differences in the overall level of methylation of the tumor cell genome, especially in the methylation of tumor suppressor genes, which can be detected at an early stage, suggesting that changes in DNA methylation may be an indicator of early detection of tumorigenesis. Tumor suppressor genes associated with HCC can be inactivated by promoter methylation, thereby stimulating tumorigenesis. 24 DNA methylation is an ideal marker for the early diagnosis of tumors due to its tissue specificity, detectability, and age independence. In addition, DNA methylation is more common compared to somatic mutations because there are more target regions and several altered CpG sites in each region of the target genome. 25 In addition to multiple CpG sites, a large number of independent hypermethylated loci in ctDNA have been identified in DBX2, THY1, MT1M, INK4A, VIM, FBLN1, and RGS10.26 Xu et al. Comparison of cfDNA samples from 1098 HCC patients and 835 healthy controls was genes associated with HCC were found to strongly correlate with the corresponding plasma cDNA methylation signatures. 25 Based on laboratory analysis, a predictive model was developed containing 10 methylation markers with sensitivity and specificity of 85.7% and 94.3%, respectively, and these markers were highly correlated with tumor mass, tumor stage, and response to treatment. These results indicate that the use of cDNA methylation markers holds great promise in the diagnosis, monitoring, and prognosis of HCC. In a methylation model consisting of three aberrantly methylated genes (APC, COX2, RASSF1A) and one miRNA (miR203) presented by Lu et al27, the sensitivity and specificity of model 27 for diagnosing HBV-associated HCC were comparable. 80%. In addition, the model could detect 75% of undiagnosed HCC patients with an AFP level of 20 ng/mL. The gene for the Ras-associated domain family 1A protein (RASSF1A) is the main repetitive DNA sequence in the human genome. Araujo et al. concluded that hypermethylation of the RASSF1A promoter could be a valuable biomarker for early screening of HCC and a potential molecular target for epigenetic therapy. 28 In one study, serum RASSF1A promoter hypermethylation was found in 73.3% of patients with HCC. 29 Long interspersed nucleotide element 1 (LINE-1) is another highly active retrotransposition mediator. Hypomethylation of LINE-1 was found in the DNA of 66.7% of HCC serum samples and was associated with early recurrence and poor survival after radical resection. 29 Hypermethylation is a common genetic process that plays a unique role in the development of liver cirrhosis and HCC. 30 In contrast, hydroxymethylation is a demethylation process that induces gene reactivation and expression, and the detection of the 5-hydroxymethylcytosine (5-hmC) product in this process can be used to identify a tumor. Methylation and hydroxymethylation of cDNA are associated with tumorigenesis and may contribute to the early screening of HCC. In a study of 2554 subjects, 31 genome-wide 5-hmCs were found in cfDNA samples, and 32 genes were identified by comparing 5-hmC sequences in HCC patients and high-risk groups such as those with chronic diseases. Diagnostic models of liver diseases. and cirrhosis. This model was superior to AFP in distinguishing HCC from non-tumor tissue.
Mutations in coding regions can lead to transcriptional abnormalities, which can lead to changes in protein sequences and ultimately cancer. Single nucleotide variants are important genomic markers for early tumor screening due to their high tissue reliability and high tumor and tissue specificity. Numerous HCC-related studies using next generation sequencing (NGS) for exome and whole genome sequencing of cancer have identified common mutated cellular genes such as TP53 and CTNNB1, as well as several including ARID1A, MLL, IRF2. The new genes, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3 and JAK1 show moderate mutation rates. Mutant gene function analysis suggests that alterations in chromatin remodeling, Wnt/β-catenin and JAK/STAT signal transduction, the P53-cell cycle pathway, epigenetic modifiers, oxidative stress pathways, the PI3K/AKT/MTOR pathway and the RAS/RAF/MAPK kinase pathway play crucial roles in HCC oncogenesis.32,33 In a study in which tumor-associated mutations were detected, Huang et al found that the frequency of tumor-associated mutations dependent on ctDNA was 19.5%, and the specificity was 90%.34 In addition, patients who experienced vascular invasion were more likely to have ctDNA mutations (P=0.041) and shorter recurrence-free survival (P<0.001). Mutant gene function analysis suggests that alterations in chromatin remodeling, Wnt/β-catenin and JAK/STAT signal transduction, the P53-cell cycle pathway, epigenetic modifiers, oxidative stress pathways, the PI3K/AKT/MTOR pathway and the RAS/RAF/ MAPK kinase pathway play crucial roles in HCC oncogenesis.32,33 In a study in which tumor-associated mutations were detected, Huang et al found that the frequency of tumor-associated mutations dependent on ctDNA was 19.5%, and the specificity was 90% .34 In addition, patients who experienced vascular invasion were more likely to have ctDNA mutations (P=0.041) and shorter recurrence-free survival (P<0.001). Mutant gene function analysis suggests that changes in chromatin remodeling, Wnt/β-catenin and JAK/STAT signaling, P53 cell cycle pathway, epigenetic modifiers, oxidative stress pathways, PI3K/AKT/MTOR pathway, and RAS/RAF/ MAPK kinase pathway plays a critical role in HCC tumorigenesis.32,33 In a study that found tumor-associated mutations, Huang et al. found that the frequency of ctDNA-dependent tumor-associated mutations was 19.5% and the specificity was 90%. .34 Кроме того, у пациентов с сосудистой инвазией чаще встречались мутации цДНК (P=0,041) и более короткая безрецидивная выживаемость (P<0,001). .34 In addition, patients with vascular invasion had more cDNA mutations (P=0.041) and a shorter disease-free survival (P<0.001). Functional analysis of the mutant genes revealed chromatin remodeling, Wnt/β-catenin and JAK/STAT signaling, the P53 cell cycle pathway, epigenetic modifiers, the oxidative stress pathway, the PI3K/AKT/MTOR pathway, and the RAS/RAF/MAPK kinase pathway play a critical role in oncogenesis of HCC. 32,33 在一项检测到肿瘤相关突变的研究中，Huang 等人发现肿瘤相关突变依赖于ctDNA 的频率为19.5%，特异性为90% .34 此外，经历血管侵犯的患者更有可能发生ctDNA 突变（P=0.041）和更短的无复发生存期（P<0.001）。 32.33 在 一 项 检测 到 相关 突变 的 研究 ， huang 等 发现 肿瘤 相关 突变 依赖于 ctdna 的 为 为 19.5% ， 特异性 为 90% .34 此外 经历 血管 侵犯 的 更 可能 发生 ctdna突变（P=0.041）和更短的无复发生存期（P<0.001）。 32,33 In a study that found tumor-associated mutations, Huang et al. found that tumor-associated mutations were 19.5% dependent on cDNA with a specificity of 90% 34. In addition, patients who had undergone vascular invasion were more likely to develop cDNA. мутация (P = 0,041) и более короткая безрецидивная выживаемость (P <0,001). mutation (P=0.041) and shorter disease-free survival (P<0.001). Another common HCC driver gene is TP53, which has a mutation rate of over 30%. Studies have shown that the frequency of TP53 mutations in ctDNA in blood and urine ranges from 5% to 60%. 35 Johan’s study showed that the ctDNA mutation spectrum in late HCC has a similar mutation rate to early HCC, including the TERT promoter (51%), TP53 (32%), CTNNB1 (17%), PTEN (8%), mutations in AXIN1 . , ARID2, KMT2D and TSC2 (6% each). 36 The β-catenin (CTNNB1) oncogene plays an important role in the Wnt signaling pathway. The transcription coactivator CTNNB1 can promote gene expression, which can lead to cell proliferation, inhibition of apoptosis, and angiogenesis. CTNNB1 can also interact with TERT to induce hepatocyte transformation. 33 The TERT promoter is frequently mutated in some solid tumors. Alterations in TERT, one of the earliest genetic changes in malignant transformation of HCC, may lead to telomerase reactivation in cirrhotic hepatocytes and may promote proliferation and prevent aging. Mutations in the 33-37 TERT promoter have been reported to occur in 59-90% of patients with proliferative liver nodules and early HCC and are associated with survival. 38
Copy number changes (CNA) are an important subtype of somatic mutations. Research has shown that the widespread and focal burden of CNA is a genomic signature capable of predicting tumor immune infiltration and exclusion in some types of cancer. 39 Active infiltration signaling, high cytolytic activity, severe inflammation and genetic markers associated with antigen presentation in HCC. Analysis of the data array of single nucleotide polymorphisms in 477 subjects revealed a low burden on the CNS. In contrast, chromosomally unstable tumors with a high extensive CNA load showed signs of immune rejection and were associated with proliferation, DNA repair, and TP53 dysfunction. Xu et al. showed that the HCC group had higher CNA scores than the chronic liver disease group. 40 Using whole-genome sequencing of a single cell, CNAs were found to appear early in hepatocarcinogenesis and remain relatively stable during tumor progression. 41 Chung et al. found that cfDNA levels were significantly elevated in HCC patients and that genome-wide CNAs in cfDNA were an important independent prognostic marker in HCC patients treated with sorafenib. 42 Patients with a higher CNA burden were more likely to have disease progression and death than those with a lower CNA burden. Ollerich et al. found that copy number instability index (CNI) can be used to assess CNA in cfDNA of cancer patients. They noted that patients with advanced cancer had significantly higher CNI scores than a control group, which assesses patient response to systemic chemotherapy and immunotherapy. 43 These results suggest that CNAs found in liquid biopsy specimens may serve as prognostic indicators in patients with advanced cancer. HCC on the background of systemic therapy.
Currently, the methods used to detect ctDNA can be divided into targeted and non-targeted methods. Briefly, targeted methods such as digital polymerase chain reaction (dPCR), BEAMing digital PCR, Amplification Refractory Mutation System-PCR, Capp-Seq and Tam-Seq are highly sensitive to predefined genes. Off-target methods such as whole genome sequencing and NGS provide a comprehensive view of the entire genomic landscape. 44 Compared to target panels, whole genome sequencing can detect not only point mutations and insertions, but also rearrangements and copy number variations. prognosis, and CTC and cfDNA are good indicators that can be used for dynamic monitoring of HCC. 45 In addition, cfDNA analysis may be more useful in detecting HCC. Yan et al. showed that cfDNA in the plasma of patients with HCC was significantly higher than in patients with liver fibrosis and healthy controls. Compared to AFP, ctDNA is expected to be a better screening marker for early HCC. 46 In a prospective study of 47 liquid biopsies that tested cfDNA and protein in a population population, they were shown to be effective in differentiating patients with HCC from patients without HCC. In a follow-up of 331 ultrasound normal and AFP-negative patients, the sensitivity and specificity of cfDNA for diagnosing HCC were 100% and 94%, respectively, so cDNA could detect HCC in asymptomatic HBsAg seropositive individuals. In the Yeo48 study, a high frequency (92.5%) of hypermethylation of the RASSF1A promoter was found in patients with HCC. In addition, Xu et al. created a diagnostic model to predict HCC using a panel of specific methylation markers with a specificity and sensitivity of 90.5% and 83.3%, respectively. The panel allows patients with HCC to be distinguished from patients with other liver diseases, which is better than AFP. They also found that normal controls that tested positive may have risk factors for HCC, such as HBV infection or a history of alcohol use. 25 We hypothesize that high-risk factors for HCC may promote hypermethylation of cfDNA, which then contributes to the progression of HCC, and thus cfDNA may play a key role in screening for high-risk groups. Cai et al. summarize the full range of ctDNA mutations and offer a robust strategy for assessing tumor burden in patients. 49 This strategy can identify tumorigenesis a median of 4.6 months before imaging change and has shown superior diagnostic performance compared to serum biomarkers AFP, AFP-L3, and PIVKA-II. The diagnostic value of cDNA testing has been demonstrated when image evaluation is not available, so cDNA testing is of value in the diagnosis of early HCC in high-risk groups. Recently, scientists used NGS technology to analyze indicators of multivariate genetic variation (including 5-hydroxymethylcytosine, 5′-motif, fragmentation, nucleosome trace, HIFI) in 3204 clinical samples and cfDNA. 50 Re-validated HIFI models with three independent train, test, and test sets showed stable and reliable discrimination between HCC and non-HCC populations with 95.79% and 95.42% sensitivity in HCC-specific test and test sets, respectively. The sexes were 95.00% and 97.83%, respectively. The diagnostic value of the HIFI method is higher than that of AFP in distinguishing HCC from cirrhosis. In addition, ctDNA is also used in surgical treatment. Atsushi et al. determined the preoperative serum levels of ctDNA in patients with HCC and found that the recurrence rate and the extrahepatic metastasis rate in the cDNA positive group were significantly higher than in the cDNA negative group, and cDNA levels were significantly correlated. with tumor progression. 51 Being a highly sensitive biomarker, ctDNA can predict the ability of HCC to invade vessels. Wang et al. performed whole genome sequencing of 46 patients with HCC, and multivariate analysis showed that the threshold value of the allele frequency of the cDNA variant for invasion into microvessels is 0.83%, sensitivity 89.7% and specificity 80.0%. an independent risk factor for microvascular invasion in resectable HCC, suggesting that cDNA may help guide optimal treatment. In conclusion, ctDNA is fully implicated in the occurrence and development of HCC and can be used for early screening, surgical evaluation, and disease monitoring.
CTCs are malignant cells derived from primary tumors or metastases that metastasize to the bloodstream. Tumor cells secrete matrix metalloproteinases (MMPs), which break down the basement membrane, allowing tumor cells to directly enter the blood and lymph vessels. However, most CTCs are rapidly eliminated by anoikis, immune attack, or shear stress. 53 The epithelial-mesenchymal transition (EMT) allows CTCs to be readily isolated from the primary tumor tissue, invade capillaries, and acquire significantly improved survival, metastasis, invasiveness, and drug resistance. Studies have shown that there is profound heterogeneity among the various tumor cells in primary metastatic tumors. Thus, CTC analysis can lead to a comprehensive understanding of tumor cell heterogeneity. 54
Specific markers for HCC-associated CTCs include glypican-3 (GPC3), asialoglycoprotein receptor (ASGPR), epithelial cell adhesion molecule (EpCAM) and stem cell-associated markers such as CD44, CD90, 55 and intercellular adhesion molecule 1 (ICAM1). ) .56 The GPC3 marker is a cell membrane-anchored protein that is clinically used for pathological analysis and characterization of HCC. 57 Expression of GPC3 is more common in HCC tumor cells with intermediate and low differentiation and promotes extrahepatic migration; in addition, the presence of GPC3+ CTCs indicates metastatic HCC. 58 ASGPR is a transmembrane protein expressed only on the surface of hepatocytes and is highly expressed in well-differentiated HCC. EpCAM is one of the most commonly used membrane-associated proteins to capture CTCs. EpCAM has been identified as a surface marker of HCC cells with stem cell characteristics,59 which correlates with various clinicopathological features of HCC, such as vascular invasion, assessed AFP levels, and advanced stage of liver cancer at the Barcelona Hospital (BCLC). The 60 CTC EMT phenotype is highly metastatic. 54 EMT processes in the CTC promote HCC metastasis. Expression of EMT markers such as vimentin, twist, E-box zinc finger binding (ZEB) 1, ZEB2, snail, slug, and E-cadherin have been studied in liver-derived CTCs from HCC patients. 58 The CanPatrol™ system developed by Cheng [61] classified CTCs into three phenotypic subgroups based on predominantly expressed markers: epithelial phenotype (EpCAM, CK8/18/19), mesenchymal phenotype (vimentin, coiled), and mixed phenotypes. In 176 patients, total CTC was superior to AFP in differentiating HCC from benign liver disease. The AUC values ​​for total CTC, AFP, and combined total CTC and AFP were 0.774 (95% CI, 0.704–0.834), 0.669 (95% CI, 0.587–0.750), and 0.821 (95% CI, 0.756–0.886). ), respectively. CTC classification based on EMT can predict HCC diagnosis, early recurrence, metastasis, and shorter overall time.
Currently, methods for detecting CSCs include physical methods and biological methods. Physical methods, often referred to as enrichment based on biophysical properties, mainly depend on the physical properties of the CSC, such as size, density, charge, mobility and deformability. Depending on the physical properties, there are various methods such as filtration-based systems, dielectrophoresis, etc. The latter, also known as immunoaffinity-based enrichment, is mainly based on antigen-antibody binding since the method uses antibodies against tumor-specific biomarkers such as as EpCAM, ASGPR, human epidermal growth factor receptor 2 (HER2), prostate specific antigen (PSA), human pancytokeratin (P-CK) and carbamoyl phosphate synthase 1 (CPS1). 62 Another type, called the no-enrichment method, uses flow cytometry to differentiate CTCs from leukocytes based on a higher nuclear-to-cytoplasmic ratio and size. Currently, the only FDA-approved test for the detection of CTCs is the Cell-Search™ system, which uses the EpCAM cell surface marker. However, combined markers-based CTC detection could increase the positivity rate.54 A mixture of antibodies against ASGPR and CPS1 achieved a CTC detection rate of 91% in HCC patients.63 Zhang et al used a CTC-Chip with antibodies against ASGPR, P-CK and CPS1, and differentiated HCC patients from those with benign liver disease or non-HCC cancer at a rate of 100%.64 A study by Wang detected EpCAM+ CTCs in 60% of 42 HCC patients and found significant correlations between both the positivity rate and the number of CTCs with TNM stage.65 Guo et al found that a CTC-derived PCR score was elevated in 125/171 (73%) patients whose AFP level was <20 ng/mL with a sensitivity of 72.5% and a specificity of 95.0%, compared with 57.0% and 90.0% for AFP at a cutoff 20 ng/mL.66 The combination of AFP and CTCs may improve HCC detection.45 It is believed that CTCs have an advantage over AFP in early screening of groups at high-risk for HCC. However, combined markers-based CTC detection could increase the positivity rate.54 A mixture of antibodies against ASGPR and CPS1 achieved a CTC detection rate of 91% in HCC patients.63 Zhang et al used a CTC-Chip with antibodies against ASGPR, P -CK and CPS1, and differentiated HCC patients from those with benign liver disease or non-HCC cancer at a rate of 100%.64 A study by Wang detected EpCAM+ CTCs in 60% of 42 HCC patients and found significant correlations between both the positivity rate and the number of CTCs with TNM stage.65 Guo et al found that a CTC-derived PCR score was elevated in 125/171 (73%) patients whose AFP level was <20 ng/mL with a sensitivity of 72.5% and a specificity of 95.0%, compared with 57.0% and 90.0% for AFP at a cutoff 20 ng/mL.66 The combination of AFP and CTCs may improve HCC detection.45 It is believed that CTCs have an advantage over AFP in early screening of groups at high risk for HCC. However, marker-based combined detection of CTCs may increase the percentage of positive results.54 A mixture of anti-ASGPR and CPS1 antibodies achieved a CTC detection rate of 91% in patients with HCC.63 Zhang et al. used a CTC-Chip with antibodies against ASGPR, P-CK and CPS1, and also distinguished patients with HCC from those with benign liver disease or non-HCC at a rate of 100%. частота и количество ЦОК со стадией TNM.65 Guo и соавторы обнаружили, что показатель ПЦР, полученный из ЦОК, был повышен у 125/171 (73%) пациентов, у которых уровень АФП был <20 нг/мл с чувствительностью 72,5% и специфичность 95,0% по сравнению с 57,0% и 90,0% для АФП при пороговом уровне 20 нг/мл.66 Комбинация АФП и ЦОК может улучшить обнаружение ГЦК.45 Считается, что ЦОК имеют преимущество перед АФП при раннем скрининге групп. frequency and number of CTCs with TNM stage.65 Guo et al found that the PCR derived from CTCs was elevated in 125/171 (73%) patients who had AFP levels <20 ng/mL with a sensitivity of 72.5% and a specificity of 95.0% compared to 57.0% and 90.0% for AFP at a cut-off level of 20 ng/mL.66 The combination of AFP and CTCs may improve the detection of HCC.45 CTCs are considered to have an advantage over AFP in early screening groups. with a high risk of HCC. However, marker-based combined detection of CTCs may increase the percentage of positive results. 54 A mixture of anti-ASGPR and CPS1 antibodies achieved a 91% CTC detection rate in patients with HCC. 63 Zhang et al. used CTC chips with antibodies against ASGPR, P-CK and CPS1 and distinguished patients with HCC from benign liver disease and non-HCC with 100%. 64 Wang’s study identified 60% of EpCAM+ CTCs in 42 HCC patients and found a significant correlation between incidence and number of CTCs at the TNM stage. 65 Guo 等人发现，在AFP 水平<20 ng/mL 的125/171 (73%) 名患者中，CTC 衍生的PCR 评分升高，敏感性为72.5%，特异性为95.0%，而AFP 在截止值为20 ng/mL 时的特异性为57.0% 和90.0%。 65 Guo 等 人 发现 在 在 在 水平 <20 ng/ml 的 125/171 (73%) 名 患者 ， ， ctc 衍生 pcr 评分 ， 敏感性 为 为 72.5%， 为 95.0%， AFP 在 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止 截止值为20ng/mL 时的特异性为57.0% 和90.0%。 65 Guo et al. обнаружили, что у 125/171 (73%) пациентов с уровнем АФП <20 нг/мл показатели ПЦР, полученные с помощью ЦОК, были повышены с чувствительностью 72,5% и специфичностью 95,0%, в то время как АФП на уровне отсечки Специфичность составляла 20 нг/мл. found that in 125/171 (73%) patients with AFP levels <20 ng/mL, CTC-derived PCR values ​​were elevated with a sensitivity of 72.5% and a specificity of 95.0%, while AFP was at cut-off specificity was 20 ng/mL. ml was 57.0% and 90.0%. 66 The combination of ORP and CTC improves the detection of HCC. 45 CTCs are thought to be superior to AFP in early screening of high-risk HCC populations. Thus, for CTC-positive and high-risk HCC groups, CTC testing should be routinely combined with ultrasound and AFP detection. However, CTCs are considered important predictors of tumor metastasis and recurrence, and detection of CTCs is not independently recommended as a diagnostic tool. 62 Therefore, CTC may serve as a better predictive biomarker than other currently used markers. Zhou et al found that patients with elevated numbers of EpCAM+ CTCs and regulatory T cells showed a higher risk of developing HCC recurrence, than those with low numbers of CTCs, with a recurrence ratio of 66.7% vs 10.3% (P < 0.001).67 A similar study was reported by Zhong et al.68 In addition, Qi found that 101 of 112 patients (90.81%) with HCC, including those with early-stage disease, were positive for CTCs and that very small HCC nodules were detected after 3 to 5 months of follow-up. Zhou et al found that patients with elevated numbers of EpCAM+ CTCs and regulatory T cells showed a higher risk of developing HCC recurrence than those with low numbers of CTCs, with a recurrence ratio of 66.7% vs 10.3% (P < 0.001).67 A similar study was reported by Zhong et al.68 In addition, Qi found that 101 of 112 patients (90.81%) with HCC, including those with early-stage disease, were positive for CTCs and that very small HCC nodules were detected after 3 to 5 months of follow-up. Чжоу и др. обнаружили, что у пациентов с повышенным количеством ЦОК EpCAM+ и регуляторных Т-клеток риск развития рецидива ГЦК был выше, чем у пациентов с низким количеством ЦОК, с коэффициентом рецидивов 66,7% против 10,3% (P <0,001)67. Zhou et al found that patients with elevated EpCAM+ CTCs and regulatory T cells had a higher risk of HCC recurrence than those with low CTCs, with a recurrence rate of 66.7% vs 10.3% (P<0.001 )67. A similar study was carried out by Zhong et al. 68. In addition, Qi found that 101 out of 112 patients (90.81%) with HCC, including those with early disease, had CTCs, and that very small HCC nodules were detected after 3 to 5 months of follow-up. Zhou 等人发现，与CTC 数量较少的患者相比，EpCAM+ CTC 和调节性T 细胞数量升高的患者发生HCC 复发的风险更高，复发率分别为66.7% 和10.3% (P < 0.001)。 Zhou 等 人 发现 与 与 ctc 数量 少 的 患者 相比 ， epcam+ ctc 和 t 细胞 数量 的 患者 发生 hcc 复发 风险 更 ， 复发率 分别 为 为 为 为 为 为 为 为 为 为 为 为 为 为 为 为 10.3% (p <0.001)。。。。。。。。。。。。。。。 Чжоу и др. обнаружили, что пациенты с повышенным количеством ЦОК EpCAM+ и регуляторных Т-клеток имели более высокий риск рецидива ГЦК по сравнению с пациентами с меньшим количеством ЦОК, с частотой рецидивов 66,7% и 10,3% соответственно (P <0,001). Zhou et al. found that patients with elevated EpCAM+ CTCs and regulatory T cells had a higher risk of HCC recurrence compared with patients with fewer CTCs, with recurrence rates of 66.7% and 10.3%, respectively (P < 0.001 ). A similar study was reported by Zhong et al. 68 In addition, Qi found that 101 of 112 HCC patients (90.81%), including patients with early disease, had positive CTC results and found very small HCC nodules after 3 visits. Observation up to 5 months. They also found CTCs in 12 patients with chronic HBV infection and found small HCC tumors within 5 months in 2 CTC-positive patients. 69 Thus, CTCs can be used to predict HCC, 70 but they can be used more routinely as predictive biomarkers.
Like cfDNA, cfRNA is released into the bloodstream through various systems. These molecules in the peripheral blood represent the cancerous tissue of origin. Compared to markers detected by non-invasive methods, cfRNAs are more dynamically regulated, tissue-specific, and abundant in the extracellular environment. The significance and diagnostic value of 71 miRNAs (miRNAs) in HCC has been reported in many studies. miRNAs are endogenous non-coding RNAs (ncRNAs) that regulate various molecular biological activities by inhibiting the translation of target messenger RNAs (mRNAs). miRNAs are located in apoptotic bodies encapsulated in exosomes, but they can also stably bind to serum proteins and lipids in peripheral blood and can be used to evaluate HCC. microRNAs are involved in liver regeneration, lipid metabolism, apoptosis, inflammation, and the development of HCC. 72 Oncogenic miRNAs such as miR-21, miR-155 and miR-221 are well known in HCC. In particular, miR-21 plays a key role in collagen synthesis in the extracellular matrix and fibrosis and promotes hepatocarcinogenesis by activating hematopoietic stem cells. 72,73 Tumor suppressor miRNAs in HCC include miRNA-122, miRNA-29, the Let-7 family, and the miRNA-15 family. The Let-7 family consists of many tumor suppressor miRNAs that target the RAS family. The miR-15 family includes miR-15a, miR-15b, miR-16, miR-195, and miR-497, which have complementary sequences for certain mRNAs. In addition, long non-coding RNAs (lncRNAs) and circular RNAs (cirRNAs) are also important for early screening of HCC. lncRNAs represent the broadest class of ncRNAs, including mRNA-like ncRNAs, and are involved in the pathogenesis of many human diseases. LncRNAs play a regulatory role in the liver microenvironment and chronic liver disease. 74 CircRNAs are also a class of ncRNAs with multiple functions in the regulation of gene expression. Recently, circRNAs have been considered as diagnostic tools for HCC.
Circulating free RNA has remarkable stability, including resistance to temperature, pH, and RNase, which makes the isolation of fnRNA from peripheral blood less tedious using standard RNA purification methods. The most commonly used methods include NGS, microarray and RT-qPCR. NGS allows microRNAs to be measured throughout the genome. However, this method is expensive and the analysis is not standardized. In contrast, RT-qPCR is inexpensive, rapidly amplifies nucleic acids, and offers many advantages such as higher sensitivity, higher accuracy, wider dynamic range, and requiring fewer samples. Microarrays are another method used for miRNA detection based on sensitive and specific hybridization of target miRNAs with complementary DNA probes, 75 but analysis of microarray data is time consuming.
Circulating miR-122 and Let-7 have been reported to be potentially useful in diagnosing early-stage HCC in high-risk groups, markers in patients with HBV-associated premalignant nodules and early-stage HCC. 76 Cai et al. found that members of the Let-7 family (miR-92, miR-122, miR-125b, miR-143, miR-192, miR-16, miR-126, and miR-199a/b) are at risk of chronic HCC in patients with hepatitis. The Let-7 family can serve as an effective surrogate biomarker for predicting the development of HCC in high-risk groups associated with chronic hepatitis C. 77 miR-122 has high diagnostic accuracy in detecting early HCC in patients with liver cirrhosis. 78 Serum circulating MiR-107 has also been evaluated in the early stages of HCC, 79 and has shown good potential in high-risk populations. Zhou et al reported that a panel of miRNAs (miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a and miR-801) can differentiate HCC from chronic hepatitis B (CHB) and cirrhosis sensitivity was 79.1% and 75%, and specificity 76.4% and 91.1%, respectively. 80 In HBV-related HCC, we found that miR150 levels were significantly reduced compared to those in chronic HBV patients without HCC (sensitivity 79.1%, specificity 76.5%). -224 was elevated in HCC compared with healthy controls, and subgroup analyzes showed higher levels in patients with HCC associated with HBV. hepatitis B-associated cirrhosis and HCC patients identified an siRNA classifier containing seven differentially expressed siRNAs that can detect HCC in different controls; AUC range at early screening is better than AFP volunteers. They found that four miRNAs (miR-1972, miR-193a-5p, miR-214-3p, and miR-365a-3p) could distinguish patients with HCC from patients without HCC. Five overexpressing miRNAs (miR-122-5p, miR-125b-5p, miR-885-5p, miR-100-5p, and miR-148a-3p) are considered potential HBV infections in HCC, cirrhosis, and CHB biomarkers, especially miR-34a-5p may be biomarkers for liver cirrhosis,85 and may be potential biomarkers for early screening of HCC in high-risk populations. The most studied lncRNA in HCC is highly activated in liver cancer (HULC). Other studies have shown that HULC circulating in HCC patients can be used as a diagnostic marker because this lncRNA is highly upregulated in HCC patients compared to healthy individuals. 71,86 Among other lnRNAs, LINC00152 is considered the best diagnostic lncRNA due to its high AUC, sensitivity and specificity. 86 In one study, peripheral blood expression of LINC00152 gradually increased from normal healthy controls to patients with CHB and cirrhosis, and finally was highest in HCC. Studies of the expression of circSMARCA5 in the plasma of patients with HCC have shown a progressive decline in expression in HCC ranging from hepatitis to cirrhosis and precancerous lesions. 87 Analysis of ROC curves confirmed the potential of these circRNAs in distinguishing patients with hepatitis or liver cirrhosis from those with HCC, especially those with AFP levels below 200 ng/mL. In addition, Zhu analyzed 13,617 cyclic RNAs in plasma samples from HBV-associated HCC patients and confirmed that 6 cyclic RNAs were expressed differently in HCC and HBV-associated cirrhosis, suggesting that cRNAs may be beneficial. markers for early screening of high-risk groups such as those associated with liver disease, sclerosis patients. 88
Exosomes are membrane vesicles 40–160 nm in diameter; multiple intracellular vesicles fuse with the cell membrane and are released into the extracellular matrix. They contain many active components, including lipids, proteins, RNA and DNA, and play a key role in communication between cells, both HCC and non-HCC cells. 89,90 Exosomes regulate the progression of HCC by activating hepatocyte fibroblasts and stellate cells, immune cells, normal hepatocytes, and HCC cells. 91 In the tumor microenvironment, tumor cells produce a large number of exosomes that are carried from cancer cells to immature cells, which in turn are involved in oncogenesis, degradation, and cellular signaling. 92 Studies have shown that exosomes can transfer oncogenes to normal cells during pathological processes, which may be one of the mechanisms of tumor invasion and metastasis. 93 The role of exosomes in cancer progression may be dynamic and specific to cancer type, 89 Exosomes may be internalized by adjacent or distant cells to regulate multiple target genes in recipient cells that may be involved in intercellular communication ions and cellular microenvironment interactions, they may mediate cellular signaling and metabolism. 94 Characteristics and dynamic changes of exosome cargo molecules directly reflect the characteristics and dynamic changes of parental tumor cells,95 which is also the basis for the use of exosomes in the diagnosis and prognosis of cancer, as well as for predicting individual response to anticancer therapy. . 96
Traditional laboratory methods for isolating and analyzing exosomes are complex, multi-step, and time-consuming, including ultracentrifugation, filtration, size exclusion chromatography, immunoaffinity purification, Western blotting, enzyme-linked immunosorbent assay (ELISA), PCR, and flow analysis. miniaturized systems and lab-on-a-chip platforms using micro/nanotechnology are being widely developed for fast, convenient in situ isolation of exosomes. Nanoparticle tracking analysis (NTA) is a widely used method for characterizing the size and concentration of exosomes, including methods such as magnetic nanoparticles and polyhydroxyalkanoates. Microfluidic and electrochemical methods can also rapidly detect exosomes in high yields.
Exosomal proteins are important markers for the diagnosis of HCC. In the Arbelaiz study, the level of 98 RasGAP SH3 binding protein (G3BP) and polymeric immunoglobulin receptor (PIGR) was significantly elevated in HCC-derived exosomes, and the putative combined efficacy of the two proteins was superior to that of AFP. Iron overload is an important factor contributing to the development of HCC. Tseng reported that hepcidin may play a key role in resistance to HCC. 99 Exosomes derived from the sera of HCC patients had a significantly higher copy number of hepcidin mRNA variants than their healthy counterparts, suggesting that hepcidin may be a novel diagnostic biomarker for HCC. The 14-3-3ζ protein in exosomes produced by 100 HCC can reduce T cell activation, proliferation, and differentiation and can induce T cell transformation into regulatory T cells, resulting in T cell depletion. 101 This is supported by several studies investigating tumor evasion from immune surveillance, 102 which may contribute to HCC tumorigenesis.
In addition to the presence of ecRNA in plasma or serum, RNA-enriched exosomes can be used for non-invasive real-time staging in early tumor screening and to determine tumor evolution and response to therapy. The level of exosomal miRNA-21 in the blood serum in the HCC group was 2.21 times higher than in the CHB group, and in the HCC group it was 5.57 times higher than in the healthy population. In the Wang study, exosomes significantly increased HCC compared with cirrhotic patients with AUC values ​​of 0.83 (95% CI 0.74–0.93) and 0.94 (95% CI 0.88–1.00). 104 The data obtained elucidated the involvement of specific exosomal cargo molecules in the regulation of oncogenesis and HCC progression. 105 Serum expression of miR-221, miR-103, miR-181c, miR-181a, miR-93 and miR-26a is consistent. and metastasis, and miR21 levels were much higher in HCC patients than in healthy controls and also in CHB patients.102 LncRNA had potential diagnostic value in HCC. Studies have shown that exosomes derived from the sera of HCC patients have significantly higher levels of LINC00161, LINC000635, and lncRNA activated by transforming growth factor-β than in patients without HCC, and these lncRNAs are strongly associated with TNM stage and tumor volume. 110 Conigliaro et al. CD90+ exosomes were found to express high levels of lncRNAH19, which significantly increased vascular endothelial growth factor (VEGF) release and VEGF-R1 receptor production, thereby stimulating angiogenesis. 93 CircRNAs are another type of exosomal ncRNAs – expressed at lower but stable levels across species, circRNAs also show specificity for cell type, tissue type, developmental stage, and regulatory activity. 111 circRNAs are diagnostic biomarkers for early and minimally invasive cancer. 112 Recent clinical trials have shown that the specificity of individual miRNAs in predicting HCC is not ideal. Therefore, complex detection using multiple assays (eg, miR-122 and miR-48a in combination with AFP) may improve the identification of early HCC and the differentiation of HCC from cirrhosis. 100
Patients with CHB and liver cirrhosis are the most common high-risk group for developing HCC. For high-risk groups, once a sustained virological response has been achieved, a cost-effective surveillance strategy based on HCC risk should be developed, and early screening is the key to improving the diagnosis and treatment of HCC with a high cost-effectiveness ratio2. . Early screening methods for cancer have many limitations: effective early screening methods have not been developed for most types of cancer, and adherence is usually low. Compared to traditional early screening methods, liquid biopsy technology has obvious advantages: ease of sampling, panrac detection, good sample reproducibility, and effective response to tumor heterogeneity. Given the cost-effectiveness of the methods associated with liquid biopsy, their use in HCC screening has not been routinely tested. Despite advances in accurate detection at the molecular level, fluid biopsy is costly to detect HCC in target patients, limiting its widespread use compared to specific imaging procedures such as ultrasound and magnetic resonance imaging. 113,114 However, a previous study showed that liquid biopsy showed a significant benefit in terms of quality-adjusted life years (QALYs). 115 The benefits of liquid biopsy in early carcinoma of the stomach and nasopharynx have also been shown. 116,117 The current view is that liquid biopsy can complement serum biomarkers and radiological screening in the detection and diagnosis of tumors. 117 118
According to current literature, fluid biopsy technology has shown significantly higher sensitivity and specificity in early screening of high-risk groups for liver cancer. Regardless of the type of fluid biopsy, it can distinguish HCC from high-risk individuals without HCC, suggesting the importance of early screening as differences between high-risk and healthy individuals are evident. ctDNA has a short half-life and can be used to detect HCC, so any changes in tumor-derived cDNA can provide real-time concrete evidence of tumor progression, especially for small tumors. A high level of ctDNA indicates the development and spread of cancer and is an early indicator of progression and recurrence. In addition, based on the results of ctDNA, patients can receive individualized treatment and follow-up. 119 Specific methylation sites may be a better marker than AFP for early identification of HCC and cirrhotic nodules. In resectable cases of HCC, high levels of cDNA are indicative of microvascular invasion and postoperative recurrence and metastasis. Changes in copy number are associated with the survival of patients with HCC. It can be assumed that cDNA evaluation may be involved in the overall treatment of HCC, and cDNA can serve as an effective indicator of therapeutic modulation. Markers based on specific genetic mutations in ctDNA have been adopted by clinical guidelines to predict efficacy and monitor drug resistance. ctDNA testing may be the most useful liquid biopsy tool for early screening. CTCs also play a key role in the early screening of high-risk HCC groups. Various markers of HCC-associated CTCs are of particular importance in the onset, development, and recurrence of HCC. As membrane vesicles, exosomes are involved in intercellular communications, especially in HCC cells. Circulating microRNAs are stable in the blood and thus may be more useful for early screening of HCC. Gradually, exosomal proteins and RNA-rich exosomes were discovered, and their predictive efficacy for HCC was confirmed. Interestingly, different etiologies of HCC may also be associated with different mutations, so we can select different biomarkers for early screening based on different etiologies of HCC. 120
However, current fluid biopsy techniques are questionable in terms of stability and cannot independently perform early screening or monitoring of HCC, but can still complement individual screening and diagnosis. 121 As a form of liquid biopsy, the detection and imaging of ctDNA, CTC, cfRNA and exosome-associated AFP or PIVKA-II have promising applications in the early diagnosis and prognosis of HCC. However, the exact mechanism of ctDNA release into the blood remains to be elucidated. Revealing the basic biological properties of ctDNA may facilitate its use as a marker. The small amount of ctDNA in the circulation and strict sample handling requirements are challenges for the clinical implementation of cDNA detection in HCC. In addition, genetic mutations do not have specific features that allow accurate identification of carcinogens. Since multiple genetic and somatic variants are also present in normal tissues, genetic mutations identified by fluid biopsy may be of limited utility in early screening for HCC. 122 The limitations of well-defined useful gene targets and biomarkers that help differentiate cDNA from non-tumor DNA are the most important issues in the use of cDNA. lack of usefulness of sensitive and specific markers for the detection of CTCs. Only viable cells with metastatic potential were found, and the optimal combination of CSC enriched markers was unclear. Isolation of CTCs for culture and evaluation of their mutational profiles is also a challenging task. Due to problems with the identification, isolation and purification of exosomes, the specific molecular mechanism is still unclear, and previous studies on the mechanism of exosomes and HCC have not been in depth, and the way miRNAs, lncRNAs, and proteins are sorted into exosomes, and it is not clear whether exosome uptake is a specific type process. The use of exosomes for the diagnosis and treatment of HCC is still at the preclinical stage. The lack of standardization of liquid biopsy procedures, such as the type of tubes used to collect blood, blood volume, sample storage and detection, isolation and enrichment, may preclude their use in routine clinical practice due to differences in practices across medical centers. The efficacy of liquid biopsy in early screening, diagnosis, efficacy evaluation, and prediction of HCC remains to be explored, especially for high-risk groups. Liquid biopsy technology has great potential and is expected to be widely used in the clinical practice of liver cancer in the near future.
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