Expert consensus on the role of hematological markers in the early clinical screening of hepatocellular carcinoma☆,☆☆,☆☆☆

Infectious diseases branch of Chinese Preventive Medicine Association

Keywords:Hepatocellular carcinoma (HCC)Early screening Hematological marker Alpha-fetoprotein (AFP)Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3)Des-gamma-carboxyprothrombin (DCP)Expert consensus

ABSTRACT The disease burden of hepatocellular carcinoma (HCC) in China is heavy, and the prognosis is still unfavourable.Therefore,early screening of high-risk groups of HCC through simple methods is the key to achieving early diagnosis and treatment and improving survival.At present,alpha-fetoprotein and other hematological tests are still the main methods in the early screening of HCC, but the sensitivity and specificity are limited, and the risk of missed diagnosis is high. In recent years, with the continuous development of science and technology, the improvement of traditional detection methods and the emergence of novel markers such as methylated deoxyribonucleic acid and microRNA have brought hope for further improving the sensitivity and specificity of early HCC screening. This consensus summarizes the research progress of traditional and new hematological test methods and puts forward expert guidance on the role of hematological markers in the early screening of HCC to provide a basis for improving the prevention and control level in China.

1. Introduction

Primary liver cancer, or liver cancer for short, is a public health problem of global concern.According to the statistics of GLOBOCAN 2020, approximately 905,000 new cases and 830,000 deaths from liver cancer were estimated. China is a country with a high incidence of liver cancer, with 410,000 new cases and 391,000 deaths per year, accounting for 45.3% and 47.1% worldwide, respectively;liver cancer is also the fifth most common malignant tumor and the second leading cause of cancer death in China.1Liver cancer mainly includes hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma, and combined HCC and cholangiocarcinoma,among which HCC accounts for 85-90%.

Despite the development in diagnosis and treatment of HCC,the prognosis remains unsatisfactory for patients with HCC in China,and the 5-year age-adjusted overall survival increased from 11.7%in 2000-2004 to 14.1% in 2010-2014.2Therefore, it is urgent to improve the prevention and treatment of HCC in China. China currently adopts a three-level HCC prevention system: (i) primary prevention, to prevent the risk factors that can lead to HCC from initially endangering the general population; (ii) secondary prevention,to reduce or delay the occurrence of HCC in patients with chronic liver disease by controlling related etiologies and risk factors and screening and monitoring based on risk stratification;(iii)and tertiary prevention, to further reduce HCC recurrence and mortality and improve overall survival in patients with HCC who have received radical treatment. The 5-year overall survival of patients with early-stage HCC (Barcelona Clinic Liver Cancer (BCLC)stage 0-A) following radical treatment could be as high as 69.0-86.2%.3However, in China, patients with HCC are mostly in the middle and late stages at diagnosis (only 3% and 30% of the patients with BCLC stage 0-A),4who are ineligible for radical treatment; therefore, for a high-risk population of HCC, early screening for secondary prevention is the key to early diagnosis and treatment as well as long-term survival.

Early screening in high-risk populations for HCC using simple and easy techniques contributes to improving screening compliance and promoting access to early screening of HCC. Blood markers such as alpha-fetoprotein (AFP) are widely used in early HCC screening; however, due to their limited sensitivity, combination with liver ultrasonography is required in the early screening and diagnosis of HCC; whereas blood markers alone are not recommended. However, according to a meta-analysis by Tzartzeva et al.5on radiography and AFP in the diagnosis of early-stage HCC,the sensitivity of AFP combined with ultrasonography for earlystage HCC was only 63%. It suggests that the sensitivity and specificity of early HCC screening with existing blood markers combined with ultrasonography remain unsatisfactory.

With the development of screening techniques,many emerging blood markers have been put forward.Compared with radiography,peripheral blood biomarker assay is more conducive to the early screening of liver cancer in clinical practice, due to its independence from a tester's experience,good repeatability,and consistent test results.

This consensus summarizes the progress of conventional and novel hematological techniques and proposes expert guidance for early diagnosis of HCC to improve the prevention and treatment of liver cancer in China.

2. Conventional blood markers commonly used in clinical practice

2.1. AFP

AFP is a glycoprotein with a relative molecular mass of 7×104,which was first discovered in the serum of patients with HCC in 1964, and remains a common biomarker in the diagnosis of HCC.High AFP is used as a diagnostic and prognostic indicator,as it has been proved to be associated with the development and progression of HCC.

Nevertheless, AFP has certain limitations as a screening biomarker. On the one hand, benign liver disease and some other cancers are also associated with elevated AFP levels; on the other hand, only approximately 60-80% of patients with HCC have elevated AFP levels. As a result,AFP as a biomarker leads to a high false-negative rate.6According to the study by Trevisani et al.7when the cutoff value of AFP of 20 μg/L (20 ng/mL) was used, the positive predictive value was 25% in the population with HCC prevalence of 5%and 61%in the population with HCC prevalence of 20%. This suggests AFP assay alone plays a limited role in the diagnosis of early-stage HCC.For this reason,AFP assay alone is not recommended as the primary screening test for HCC, and more direct methods and biomarkers are needed to significantly improve the diagnosis of HCC.

2.2. Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3)

AFPs vary in the contained glycosyl groups;among them,AFP-L3 has the capacity of binding to lectins and is more specific than AFP in patients with HCC. In the study by Kudo et al.8AFP-L3 had a sensitivity of 18.8% and a specificity of 99.4% in the diagnosis of HCC;its insufficient sensitivity might lead to limited application in early screening of HCC. However, this study also showed that in patients with advanced HCC, the survival rate of the AFP-L3 negative and negative-to-positive groups was higher than that of the positive and positive-to-negative groups, indicating that AFP-L3 played a certain role in the diagnosis and prognosis evaluation of advanced HCC.In a study by Choi et al.9on the evaluation of three blood biomarkers for early HCC diagnosis, the combination of AFP and AFP-L3 could significantly improve the sensitivity and specificity of early HCC screening, leading to an increase of sensitivity from 62% (AFP alone) to 79% without compromising specificity(87%). However, AFP-L3 alone, with a sensitivity of 55%, is of little significance in HCC screening.

2.3. Des-gamma-carboxyprothrombin (DCP, also known as PIVKAII)

Prothrombin is a clotting protein synthesized in the liver. In patients with liver diseases, including HCC, vitamin K-dependent gamma-carboxylation is impaired and prothrombin synthesis is reduced to form DCP. In 1984, Liebman et al.10tested the serum PIVKA-II level of patients with HCC using radioimmunoassay and found that the PIVKA-II level significantly increased in 91% of patients.PIVKA-II has been included as an adjuvant diagnostic marker for HCC in the Standardization for Diagnosis and Treatment of Hepatocellular Carcinoma (2011 Version) issued by the National Health Commission of the People's Republic of China.PIVKA-II has been approved by the USA Food and Drug Administration(FDA)for risk stratification of HCC,but not for HCC monitoring.Loglio et al.11found in the screening and prediction of patients with HCC using AFP and PIVKA-II that in the 6-18 months prior to the diagnosis of HCC, the serum PIVKA-II value of 53% of patients was above the determined cutoff value(82 mAU/mL),with an accuracy of 86%.In the combination of AFP,the accuracy was further improved to 90%.The combination of AFP and PIVKA-II showed good sensitivity for the screening of HCC. However, the authors also pointed out that the baseline of DCP was also different because of the ethnic and regional differences, and the current studies cannot solve this difference, which brings certain difficulties for it to become an early diagnostic biomarker.

Recommendation 1: (i) For populations at risk of HCC, AFP is recommended as a routine blood marker for early screening of HCC,which should be combined with abdominal ultrasonography. The detection frequency is determined according to the risk stratification of target population:once a year for nonhigh-risk populations,once every 6 months for high-risk populations, and once every 3 months for extremely high-risk populations. Dynamic contrastenhanced computed tomography (CT)/magnetic resonance imaging (MRI) is recommended for clinical diagnosis if AFP is positive(duration ≥400 ng/mL), and chronic or active hepatitis, cirrhosis,embryonal tumors of the testis or ovary, and pregnancy are excluded. (ii) The combination of AFP-L3 and DCP in patients with or without increased AFP titer enhances the detection rate of earlystage HCC(reference cutoff values:AFP ≥200 ng/mL,AFP-L3 ≥15%,and DCP ≥40 mAU/mL).

3. Other blood markers

3.1. Gamma-glutamyl transpeptidase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST)

GGT is a membrane-bound enzyme, which is essential for glutathione synthesis. GGT in serum mainly comes from the liver and abnormally increases in patients with HCC. Simultaneously,GGT levels also abnormally increase in other liver diseases such as viral hepatitis, alcoholic hepatitis, and liver cirrhosis. Therefore,GGT cannot be used as an effective indicator for screening HCC,and its specificity is only approximately 30%.12ALT and AST are enzymes involved in human protein metabolism. In patients with hepatic impairment caused by various causes such as acute and chronic viral hepatitis, alcoholic hepatitis, and liver cirrhosis, the ALT and AST levels increase to varying degrees, whereas in patients with HCC, ALT and AST levels do not significantly increase or even decrease.13,14

The diagnostic value of GGT/AST ratio in hepatitis B virus(HBV)-related HCC have been studied by the Affiliated Hospital of North Sichuan Medical College.15It showed that when PIVKA-II and AFP were combined with GGT/AST ratio for diagnosis of early-stage HCC, compared with PIVKA-II and AFP alone, the area under the receiver operating characteristic curves(AUROCs)increased,which were 0.857 vs. 0.835 and 0.757 vs. 0.621,respectively, proving that PIVKA-II and AFP combined with GGT/AST ratio could improve the early diagnostic value of these tumor markers in HCC.

3.2. Glypican-3 (GPC-3)

GPC-3 is a member of the glypican family anchoring heparin sulfate proteoglycan on the cell surface with glycosylphosphatidylinositol, which is low expressed in normal human tissues, but it is found that this protein is overexpressed in the diseased liver, especially HCC.16A recent Meta-analysis by Zhao et al.17on Golgi protein 73 (GP73), GPC-3, and AFP in the early diagnosis of HCC found that the AUROC detected jointly by the three biomarkers was 0.95,indicating that the diagnostic accuracy rate by the joint detection was higher than that by GP73,GPC-3,and AFP alone or two-joint detection.A study by Di Tommaso et al.18on the diagnostic value of heat shock protein 70 (HSP70), GPC-3, and glutamine synthetase in liver cirrhosis hepatocellular nodules found that GPC-3 had a sensitivity of 68.75%,a specificity of 90.91%,a positive predictive value of 91.67%,and a negative predictive value of 66.67% in the diagnosis of early-stage HCC.

3.3. GP73

GP73 is a membrane protein, which is commonly expressed in epithelial cells of many human tissues and can also be detected in the serum of patients with liver disease. The sensitivity and specificity of serum GP73 in the diagnosis of HCC were 69-95% and 70-93%,respectively,which were significantly higher than those of AFP.Additionally,there was no significant difference in serum GP73 levels between patients with AFP-positive HCC and patients with AFP-negative HCC.19A recent study by Xiao et al.20on GP73 and AFP in the diagnosis of HCC found that although compared with the healthy control group the GP73 level in patients with HCC and chronic liver diseases(such as chronic hepatitis and liver cirrhosis)significantly increased, there was no significant difference in the GP73 levels in chronic hepatitis, liver cirrhosis, and HCC. This is a small-sample retrospective study, which needs to be further validated by a large prospective study.

3.4. Serum laminin

Laminin is a large heterotrimeric glycoprotein and an important component of normal tissue epithelium.21Laminin gamma2 is a subunit of the heterotrimeric basement membrane protein,laminin-332(composed of the alpha3,beta3,and gamma2 chains).The gamma2 chain has been highly expressed before cancer invasion, and such expression is associated with a poor prognosis of cancer,in which the gamma2 chain is expressed in monomer form in the invaded cancer cells. A recent study by Yamashita et al.22revealed that the sensitivity and specificity of laminin in the diagnosis of HCC were 62.9% and 70.5%, respectively. Additionally, patients with HCC with increased laminin gene had a poor prognosis and were prone to distant organ metastasis in the late stage.Elevated serum laminin is associated with a high incidence of HCC in patients with chronic hepatitis C who have sustained virological response to antiviral therapy.

Recommendation 2: Other serum protein markers can also be used as reference indicators and are not routinely recommended.

4. Novel blood markers

4.1. Methylated deoxyribonucleic acid (DNA)

DNA methylation refers to the covalent binding of a methyl group at the carbon position of cytosine 5 of genomic CpG dinucleotides under the action of DNA methyltransferase. DNA methylation,noncoding DNA,and histone acetylation are generally considered the main epigenetic modifications affecting gene expression. Among them, DNA methylation participates in gene transcription regulation and maintains genome stability, which is also one of the earliest discovered ways of DNA modification. In recent years, several studies using whole-genome technology to detect DNA methylation in cancer have shown that a large number of genes show an abnormal DNA methylation profile in cancer.23DNA methylation is an early event in the occurrence of HCC,including increased methylation levels of tumor suppressor genes or decreased methylation of proto-oncogenes, and this change is earlier than the mutation of driver genes. Therefore, the understanding of DNA methylation mechanism helps to clarify the pathogenesis of HCC and is helpful for the prediction, diagnosis,and monitoring of HCC.

Recently, Kisiel et al.24expanded the list of candidate methylated DNA markers (MDMs) of gastrointestinal tumors and hepatobiliary cancers by applying sequencing technology and proved the feasibility of this method in phase I clinical application. When comparing the cases with the control group,they found that there was a significant difference in RNA levels at the 11/12 region.C-type lectin domain family 11 member A (CLEC11A) and testis-specific protein Y-encoded-like 5 (TSPYL5) were significantly downregulated in tumors; whereas acid phosphatase 1 (ACP1), DOC2/DAB2 interactive protein(DAB2IP),endothelin-converting enzyme-1(ECE-1),ephrin B2(EFNB2),empty spiracles homeobox 1(EMX1),homeobox A1 (HOXA1), phosphofructokinase platelet (PFKP),serine protease inhibitor Kunitz types 2 (SPINT2), and secernin-1(SCRN1) were up-regulated. These novel MDMs had high accuracy for the detection of HCC. Simultaneously, they also compared the MDMs methylated in the liver tissues of patients with HCC and control liver tissues,but not methylated in white blood cells,which may indicate conditions such as HCC, vascular invasion, or hepatocyte death.

In another study conducted by Peking Union Medical College Hospital on secreted phosphoprotein 1 (SPP1) and lecithincholesterol acyltransferase (LCAT) as diagnostic biomarkers of serum samples from patients with HCC,25they found that SPP1 was not expressed in hepatocytes under normal conditions but showed hypomethylation and high expression in HCC. Overexpression of SPP1 was related to early recurrence, intrahepatic metastasis, and poor prognosis of HCC. Under normal circumstances, LCAT produces cholesterol ester from high-density lipoprotein in the circulation and transfers it to apolipoprotein b with the help of lipid transfer protein. With the progression of HCC injury, the LCAT activity in plasma decreases, and LCAT shows hypermethylation and low expression in HCC,and the low expression of LCAT is related to poor prognosis. The predictive model constructed by SPP1 and LCAT has predictive value for HCC with few test genes and low cost,and is also of some significance for other tumors.

4.2. Circulating cell-free DNA (cfDNA)

cfDNA is a DNA fragment released by cells in blood. At present,many trials believe that cfDNA contains relevant gene characteristics for specific tumors. Therefore, cfDNA assay may further improve the sensitivity and specificity of early diagnosis of HCC.Circulating tumor DNA (ctDNA) is a specific tumor-related DNA fragment that is secreted and released outside the cell and enters the peripheral blood during the process of renewal, necrosis, and apoptosis of tumor cells, which can reflect the cytogenetics and epigenetics information of tumor.

The concentration of ctDNA in peripheral blood fluctuates greatly,and there will be great differences among different tumors,different patients with the same tumor, and different stages of onset and treatment of the same patient. In the whole cycle management of HCC (early screening, perioperative monitoring, molecular typing, targeted medication guidance, immunotherapy medication guidance, efficacy monitoring, recurrence monitoring,prognosis prediction, etc.), ctDNA monitoring can dynamically provide genetic variation information of tumors, including point mutation,insertion/deletion,copy number variation,and structural variation, as well as epigenetic variation information, such as methylation level variation.These assay targets are all hot topics of studies in recent years.26,27The M2P-HCC (methylation, mutation,and protein) model currently under study in China is an early screening model of HCC based on ctDNA technology.The M2P-HCC model is derived from the HIT study,which was the only open-data prospective large-scale cohort study of current ctDNA liquid biopsy for early screening of HCC in China, with the Breakthrough Device Designation HCCscreen™product granted by FDA as a screening means for HCC high-risk population. HCCscreen™performs early screening for HCC using a combination of multi-omic liquid biopsy techniques that simultaneously cover comprehensive indicators including methylation of HCC-specific target genes,high frequency somatic mutations, and proteins. According to information disclosed in February 2021,the HIT study completed follow-up data for 1615 patients positive for HBsAg.The results showed that the M2PHCC model achieved 88% sensitivity and 93% specificity for HCC screening, and its screening performance was superior to that of AFP combined with ultrasonography.28In the absence of abnormal radiological data, the M2P-HCC model has detective value in detecting early-stage HCC,and it is expected to become a detection technique superior to AFP throughout the process of early-stage HCC screening and monitoring HCC diagnosis and treatment.

5-Hydroxymethylcytosine (5-hmC), a substance produced by the oxidation of 5-methylcytosine with 10-11 translocase, is involved in the construction of mammalian promoters, generegulating sequences, etc., which have also been found to be related to the modification of 5-hmC in cancer expression. In the study of Cai et al.29of Fudan University in Shanghai on 5-hmC in circulating free DNA as a noninvasive method for early detection of HCC, genome-wide 5-hmC analysis obtained from cfDNA samples of 2554 Chinese subjects found that the sensitivity and specificity for distinguishing patients with early-stage HCC from non-HCC patients were 89.6% and 78.9%, respectively, cutoff point = 27.9,which was significantly superior to the AFP-based model,and it still had a good diagnostic efficacy for patients with AFP-negative HCC,for example,the area under curve(AUC)for diagnosing early-stage HCC with AFP ＜20 ng/mL was 92.4%. This study provides a new possibility for the detection and treatment of early-stage HCC.

4.3. MicroRNA (miRNA)

miRNA is a noncoding small RNA with a length of 22-24 nucleotides,which regulates the posttranscriptional gene expression.Previous literature reported that serum miRNA was mainly present in the exosomes formed by phospholipid membrane,thus avoiding the degradation of RNase in circulation.30Therefore, it can be specifically and stably detected in blood or tissues,which makes it an ideal potential tumor marker.

In a recent study conducted by Suzhou University on miRNA as the biomarker of HCC,31they found that the expression levels of miRNA-378 in blood and tissues of patients with HCC were significantly lower than those in the control group among six candidate miRNAs. The decreased expression was related to promoter hypermethylation, and the gene variations of miRNA-378a-3p precursor RNA were positively correlated with the risk and prognosis of HBV-related HCC. Additionally, other studies on miRNA have also pointed out that miRNA-221-3p and miRNA-101-3p play an important role during tumorigenesis. MiRNA-221-3p is overexpressed in HCC tissues and serum and high expression of miRNA-221-3p in serum is related to tumor size,tumor stage,and poor prognosis.The down-regulation of miRNA-101-3p is related to invasiveness and poor prognosis of HCC, and low expression of miRNA-101-3p in plasma can predict poor disease-free survival.Another study conducted by Shandong Binzhou Medical University on miRNA-1277 as a biomarker of HCC found that miRNA-1277 was significantly down-regulated in the serum of patients with HCC and HepG2 cells.32Inhibition of miRNA-1277 can promote the proliferation, migration, and invasion of HepG2 cells, whereas overexpression of miRNA-1277 is the opposite. The study also found that as one of the transforming growth factor-β superfamily members, bone morphogenetic protein 4 (BMP4), was a direct target of miRNA-1277, which could reverse the effects of miRNA-1277 on cell migration and invasion. The expression of BMP4 was up-regulated in HCC samples, and inhibition of BMP4 could significantly reduce the migration and invasion potential of HCC.Furthermore, up-regulation of BMP4 was proved to be an important indicator for predicting the poor prognosis of patients with HCC.

A study on the mechanism of miRNA-224 in the occurrence and development of HCC as well as its diagnostic and prognostic values in the Henan Medical School showed that the expression level of serum miRNA-224 in patients with HCC was significantly higher than that in the healthy control group.33Additionally, serum miRNA-224 expression was higher in patients with larger tumor and later stage. It was also found that the higher the expression level of serum miRNA-224, the shorter the overall survival of patients, suggesting that serum miRNA-224 could be used as a prognostic factor for patients with HCC. However, this study only conducted a control study on patients with HCC and healthy people. The changes of serum miRNA-224 in patients with HCC and patients with other liver diseases still need to be further studied.

In a study conducted by Professor YuTian Chong's team from the Third Affiliated Hospital of Sun Yat-sen University, on miRNA classification for early diagnosis of HCC,data from healthy controls,inactive HBsAg carriers, patients with chronic hepatitis B, patients with hepatitis B-induced cirrhosis, and patients with HCC diagnosed in four hospitals in China were compared and a miRNA classifier consisting of seven differentially expressed miRNAs(miR-29a,miR-29c,miR-133a,miR-143,miR-145,miR-192,and miR-505)was identified. Its sensitivity for detecting HCC was higher than that of AFP,whereas the specificity was similar to that of AFP,AUC for small-sized and early-stage HCC was higher than that of AFP,and AFP-negative HCC could be detected.34

4.4. Long noncoding RNAs (lncRNAs)

LncRNAs refer to RNAs with a length of more than 200 nucleotides.Although it does not code proteins,it is related to a variety of functions,including the regulation of cis-or trans-transcription,the regulation of messenger RNA processing,and the control of protein activity after translation. Many lncRNAs are related to human diseases,and changing the expression of lncRNAs can promote cancer phenotype by stimulating cell proliferation,angiogenesis,immune evasion, metastasis and inhibiting apoptosis. A recent study conducted by Kim et al.35on serum small extracellular vesicle-derived LINC00853 as a new marker for diagnosis of early-stage HCC found that LINC00853, a member of the lncRNA family, was significantly expressed in patients with HCC. In early-stage HCC, AFP was positive in only 9% of early-stage HCC cases, whereas LINC00853 was positive in 94% of cases. Additionally, LINC00853 was positive in 97% of AFP-negative cases of early-stage HCC. These results suggested that LINC00853 was superior to AFP in the diagnosis of early-stage HCC. Moreover, LINC00853 was also highly positive even in AFP-negative early-stage HCC cases, suggesting that LINC00853 might have special value in patients with AFP-negative tumor.

4.5. Circular RNA (circRNA)

CircRNA is an exceptionally stable RNA molecule,in which the 3′tail of an exon is connected to the 5′head of the upstream exon.At present,the known functions of circRNA include transcription and splicing regulation. However, the molecular and biological functions of most circRNAs remain unknown. Recently, a study conducted by the Eastern Hepatobiliary Surgery Institute, Second Military Medical University, on the specific effect of noncoding regulatory RNA network driven by circular-chromodomain Y like(Circ-CDYL) on early-stage HCC found that Circ-CDYL was highly expressed in the early-stage of HCC and overexpression of Circ-CDYL in HCC cells could significantly promote the malignant proliferation, self-renewal, chemotherapy resistance, and tumorigenesis characteristics of HCC cells.36For early-stage HCC, when the Circ-CDYL,HDGF,and HIF1AN levels were analyzed in combination,the sensitivity and specificity were 75.36%and 66.67%,respectively,and the diagnostic efficacy was higher than that of AFP, but AFP showed higher diagnostic efficacy in advanced HCC. This study provides a new possibility for early screening of HCC.

4.6. Glycomics

Several studies have found that the patients who progress from liver fibrosis or cirrhosis to HCC show changes in biological signals of serum N-glycomics, suggesting the potential value of biological signals of serum N-glycomic as new biomarkers for HCC. Academician Hui Zhuang's team analyzed the serum N-glycomics of the patients with very early/early-stage and middle/late stage HCC and liver fibrosis/cirrhosis among 247 patients with HBV-related liver diseases and found that the sensitivity and specificity of the early diagnosis model, glycomics-very early/early-stage HCC (EHCC),established by biological signals of serum N-glycomics for detecting EHCC in liver fibrosis/cirrhosis patients was 84.6% and 85.0%,respectively, which were superior to the conventional blood marker, AFP. Additionally, the glycomics-EHCC model can detect potential EHCC 6 or even 12 months before clinical diagnosis.Glycomics analysis may become a new development direction in the future.37

Recommendation 3: For clinically undiagnosed high-risk or extremely high-risk populations with repeated AFP fluctuation but no nodule in ultrasonography or with negative AFP but with nodule in ultrasonography, the commercial novel blood marker detection kit can be used as a means of supplementary detection.

5. Conclusions

Significant progress has been made in the field of early HCC diagnosis. Conventional tumor markers such as AFP, AFP-L3, and DCP are not easily degraded in vitro; although extraction and detection techniques are relatively simple, such markers have certain limitations, as they are interfered by other diseases and cannot be used for etiological analysis of early-stage HCC;combination with abdominal ultrasonography is required in routine early screening diagnosis. However, the detection techniques based on novel blood markers such as cfDNA, miRNA, and lncRNAs still need to be validated in large-size populations in terms of sensitivity,specificity,or stability and cannot be used alone;such markers can be used as a supplement to individualized screening,monitoring or diagnosis to screen out high-risk patients with early/very early-stage HCC. The rational use of various blood markers is the key to currently improve the detection rate of early-stage HCC,and the development and popularization of blood markers with higher sensitivity and specificity are also clinical problems to be solved in the future.

Authors’ contributions

Chan Xie and Zhiliang Gao(The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China) were the writing authors of the manuscript. Zhiliang Gao (email address:gaozhl@mail.sysu.edu.cn) was the corresponding author. All authors read and approved the final manuscript.

List of experts

Xiaoguang Dou (Shengjing Hospital of China Medical University), Bingliang Lin (The Third Affiliated Hospital of Sun Yat-sen University), Feng Lin (Hainan General Hospital), Qing Mao (The First Affiliated Hospital of the Army Medical University), Junqi Niu(The First Hospital of Jilin University), Hong Ren (The Second Affiliated Hospital of Chongqing Medical University), Hong Tang(West China Hospital, Sichuan University), Lai Wei (Beijing Tsinghua Changgung Hospital), Dazhi Zhang (The Second Affiliated Hospital of Chongqing Medical University), Hui Zhuang (Peking University Health Science Center),Hongfei Zhang(Beijing Tsinghua Changgung Hospital),Wenhong Zhang(Huashan Hospital Affiliated to Fudan University), Yingren Zhao(The First Affiliated Hospital of Xi'an Jiaotong University).

Declaration of competing interest

The authors declare that they have no conflicts of interest.