What tests should be done for primary liver cancer? A complete list of methods for testing primary liver cancer

1. Serology

(1) AFp: AFp is currently the most specific marker for diagnosing hepatocellular carcinoma. AFp is an embryonic protein synthesized by the liver during the fetal period, and can regain this function when adult liver cells become malignant. Since embryonic carcinoma of the gonads can also occur in pregnant women, newborns, and testicles or ovaries, AFp has only a relatively specific diagnostic value for hepatocellular carcinoma. Due to the improvement in the sensitivity of the detection method, low concentrations of AFp can also be detected in some hepatitis, cirrhosis, and a few digestive tract cancers such as gastric cancer, colon cancer, pancreatic cancer and other metastatic liver cancers. Therefore, the AFp test results must be linked to clinical practice to be diagnostically meaningful.

At present, the radioimmunoassay (RIA) or AFp monoclonal antibody enzyme immunoassay (EIA) rapid assay is mostly used to detect serum AFp content. There is no trace amount in normal human serum, less than 20μg/L. 70-90% of patients with hepatocellular carcinoma have elevated AFp levels. Usually, AFp concentration is related to tumor size, but there are large individual differences. It is generally believed that AFp is often low or undetectable in patients with pathological differentiation close to normal hepatocytes or with extremely low differentiation. The recognized standards abroad are often high and easy to miss. my country attaches importance to the dynamic observation of increased medium and low concentrations of AFp. In clinical practice, patients with low AFp concentrations often need to be followed up with imaging diagnostic technology to help establish a diagnosis early. Liver cancer often occurs on the basis of chronic active liver disease, so it must be differentiated. Chronic hepatitis and post-hepatitis cirrhosis have increased AFp in 19.9% ​​to 44.6% of patients, with concentrations mostly between 25 and 200 μg/L. Benign liver disease activity often precedes a significant increase in alanine aminotransferase, and AFp is associated or synchronized, first high and then low. Generally, within 1 to 2 months, as the condition improves, the aminotransferase decreases, and AFp decreases accordingly, showing a "transient" state. Sometimes, AFp in benign liver disease activity may also show dynamic changes such as repeated fluctuations and continuous low concentrations, but one must be alert to the possibility of early cancer while liver disease activity is present.

⑵ Detection of other liver cancer markers: In recent years, the number of primary liver cancers with negative serum AFp has increased. Therefore, the development of newer, more specific and more sensitive markers has become an urgent task. Finding isoenzymes and heteroplasms with carcinoembryonic characteristics and finding specific subcomponents are the current research directions of liver cancer serum markers. In recent years, the following have been reported to be of high value in the diagnosis of liver cancer:

① r-GT isoenzyme (GGTⅡ): polyacrylamide gradient electrophoresis separation method can show 12 isoenzyme bands. Bands Ⅰ´, Ⅱ, Ⅱ´ are specific bands for primary liver cancer, with a positive rate of 79.7%. The positive rate of this enzyme in AFp-negative patients is 72.7%.

② Alpha-fetoprotein heterogeneity (FucAFp): Currently, the diagnostic value of AFp heterogeneity is high by using lentil agglutinin (LCA) affinity cross-immunoautography. There are two heterogeneities, namely LCA non-binding type (AFp-NL) and binding type (AFp-RL). The average AFp-NL content in liver cancer is 49.13±27.20% (0-100%), and 75% is the diagnostic standard for liver cancer. The positive rate is 86.0%, which decreases with the deterioration of the disease. The AFp-NL of non-cancerous liver disease is 93.30±7.66%, and the false positive rate is 1.6%.

③ Abnormal prothrombin: The liver synthesizes an inactive precursor of prothrombin, which is carboxylated to an active form by vitamin K, r. In liver cancer, the vitamin K-dependent carboxylation system in the microsomes of liver cancer cells is dysfunctional, and the activity of hydroxylase decreases, resulting in incomplete glutamate carboxylation, thereby forming abnormal prothrombin. Recently, it has been discovered that liver cancer cells have the ability to synthesize and release abnormal prothrombin. In China, the standard for determining abnormal prothrombin by radioimmunoautography is ≥250μg/L. The positive rate for liver cancer is 69.4%, the positive rates for liver cancer with low AFp concentration and AFp-negative liver cancer are 68.3% and 65.5%, respectively, and the compliance rate for small liver cancer is 62.2%. Most data show that abnormal prothrombin has a high specificity for primary liver cancer, and the false positive rates for various non-cancerous liver diseases, secondary liver cancer and benign liver tumors are extremely low, and it may become a valuable liver cancer marker.

④ Serum fucosidase (AFu): AFu belongs to the lysosomal acid hydrolase class, and its main physiological function is to participate in the degradation of bioactive macromolecules such as fucosylated glycoproteins and glycolipids. Primary liver cancer should be considered when AFu exceeds 110Kat/L. Domestic reports show that the positive rate of AFu in diagnosing primary liver cancer is 81.2%, and the positive rates for AFp-negative liver cancer and small liver cancer are 76.1% and 70.8% respectively. Secondary liver cancer and benign liver space-occupying lesions are negative, but the false positive rate of cirrhosis and chronic hepatitis is high.

⑤M2 pyruvate kinase (M2-pyK): Pyruvate kinase (pyK) is a key enzyme in glycolysis. There are four isoenzymes, L, R, M1M2 (k). M2 (K) is the main isoenzyme in fetal liver and liver cancer tissues, which can be regarded as a carcinoembryonic protein. The ELIS sandwich method can detect pg-level trace amounts of cancer markers with high sensitivity. The normal value is 575.8±259.5ng/L. Liver cancer is 5 times higher than normal, and it is significantly increased in the small liver cancer stage. The poorer the differentiation, the more obvious the M2-pyK value. The positive rate is 5.2%, and it can also be increased in digestive tract tumors, but not in hepatitis and benign liver tumors.

⑥ Isoferritin (AIF): Isoferritin has a certain significance in the diagnosis of liver cancer because the synthesis of liver cancer cells increases and the release rate accelerates. The normal value is 16-210μg/L, with 300μg/L as the diagnostic threshold. 72.1% of liver cancer patients exceed this value, and the false positive rate is 10.3%. The positive rate of liver cancer with negative AFp or low concentration AFp is 66.6%, and the positive rate of small liver cancer of & 5 cm is 62.5%.

⑦α-antitrypsin (AAT): Human liver cancer cells have the function of synthesizing and secreting AAT, which increases when the tumor is combined with cell necrosis and inflammation. Immunoperoxidase technology shows that 74.9% of liver cancer cases are higher than 4000ng/L, while benign liver disease is 3-10.9%. The positive rate of AFp-negative liver cancer is 22.7%.

⑧Aldolase isoenzyme A (ALD-A): When ALD-A appears and increases to >800ng/ml in liver cancer, it is helpful for diagnosis. The positive rate of AFp-negative liver cancer is 73.6%.

In summary, the above liver cancer markers have auxiliary significance for the diagnosis of primary liver cancer, especially AFp-negative cases, but they still cannot replace AFp in the diagnosis of liver cancer. According to practical experience, combined detection is better than single detection. Serum AFp detection combined with 1-2 liver cancer markers can significantly increase the positive detection rate of primary liver cancer. In clinical analysis, comprehensive judgment should be combined with medical history, imaging diagnosis or histological data to draw accurate conclusions.

2. Liver cancer imaging diagnostic examination:

⑴ Real-time ultrasound imaging (US): Ultrasound imaging is widely used in clinical practice for its high sensitivity in showing lesions of solid soft tissue organs and its low impact on human tissues, as well as its low cost. As small liver cancer gradually grows, ultrasound imaging shows that the internal echo changes from low echo to high echo and mixed echo. Tumors with a diameter of less than 2 cm often have low echo nodules; those with a diameter of 2 to 3 cm show low echoes with the same frequency as the surrounding echoes; those with a diameter of 3 to 5 cm are mostly peripheral low echoes; and those with a diameter of more than 5 cm are mostly high echoes or mixed echoes. In addition to the above-mentioned polymorphic and variable characteristics, hepatocellular carcinoma also has the following characteristics as the tumor grows: ① Halo has a clear tumor capsule, the center of the nodule is relatively uniformly high echo, and the adjacent capsule is a low echo dark ring called "halo", which is a fibrous capsule or interpreted as blood vessels around the tumor. ② Nodules in nodules: Nodules with different echoes in the high echo tumor area indicate new tumor foci growing in hepatocellular carcinoma. In addition to locating liver cancer, ultrasound imaging can also show whether there is cancer thrombus formation in the portal vein and its branches, understand the anatomical relationship between the tumor and the large blood vessels, and whether there is cancer dissemination and intra-abdominal lymph node metastasis. It is of great value in determining the treatment plan before surgery, estimating the possibility of resection, selecting the indications for hepatic artery embolization, and monitoring recurrence after surgery.

In recent years, color Doppler blood flow imaging has been widely used in clinical practice. In addition to showing space-occupying lesions, it can also measure the blood flow in and out of the tumor to identify the blood supply of the space-occupying lesion and infer the nature of the tumor. Ultrasound-guided puncture biopsy and local injection into the tumor have been widely used in the diagnosis and treatment of small liver cancer. High-resolution intraoperative ultrasound imaging can accurately locate the tumor to improve the surgical resection rate.

⑵Computed tomography (CT) Among various imaging examinations, CT can best reflect the pathological morphology of the liver, such as the size, shape, location, number of lesions, and the presence or absence of hemorrhage and necrosis within the lesions. The invasiveness of the lesions can be understood from the edge of the lesions, and the invasion of the portal vein can be understood from the cancer thrombus and invasion of the portal vein. CT is considered to be the preferred non-invasive diagnostic method to supplement ultrasound imaging to estimate the range of lesions. CT manifestations of liver cancer, plain scan manifestations: lesions are generally low-density, lower than the density of the surrounding liver parenchyma, and some lesions are surrounded by a layer of lower-density ring shadows (halo sign). The nodular type has clearer edges, while the massive and mixed types have more blurred and partially clear edges. Enhanced manifestations: After intravenous injection of iodinated contrast agents, the density of lesions and liver tissues is increased to a certain extent, which is called enhancement. Including: ① Dynamic enhanced scanning; dynamic scanning with bolus injection or rapid spiral CT scanning. In the early stage (hepatic artery stage), the lesion is enhanced with high density, which is higher than the surrounding normal liver tissue for 10 to 30 seconds. Then the density of the lesion decreases rapidly and approaches the normal liver tissue with equal density. It is easy to miss at this stage; the density of the lesion continues to decrease and the liver tissue becomes low-density. This stage can last for several minutes. The early enhanced image of dynamic scanning can easily find satellite lesions with a diameter of less than 1 cm or 1 to 2 cm, and also help to find small lesions. ② Non-dynamic scanning: ordinary scanning takes at least 15 seconds each time, so the liver layer where the lesion is located may fall in any of the above dynamic scanning stages and show different densities. Most of the lesions fall in the low-density stage, so the lesions are significantly lower than those in plain scanning. Manifestations of invasion of the portal vein system and other systems: The rate of cancer thrombosis in the portal vein system of primary liver cancer is high. The enhancement is long and the difference between the unenhanced cancer thrombosis and the obviously enhanced blood is large. The strip filling defect causes irregular or non-developed images of the portal vein trunk or branch vessels. A small number of patients have cancer thrombosis in the inferior vena cava. Invasion of the liver portal can cause dilatation of the intrahepatic bile duct, and occasionally retroperitoneal lymphadenopathy, ascites, etc. Pulmonary metastasis may appear abnormal on chest CT examination, which is more sensitive than chest X-ray.

In recent years, new CT machines have been continuously updated, and CT examination technology has been continuously improved, especially the combination of angiography and CT, such as direct injection of contrast agent into the hepatic artery for CT enhancement CTA (CT-Angiography), injection of contrast agent into the superior mesenteric artery or splenic artery during the portal venous phase for CT tomography (CTAp), and Lipiodol-ct (Lp-CT) after injection of iodized oil into the hepatic artery during angiography and CT plain scan at intervals of 2 to 3 weeks. These methods have a better detection rate for small liver cancer, especially small liver cancers of less than 1 cm, than CT dynamic scanning. However, among the above methods, CT plain scan plus enhancement is still the routine, and CTA and CTAp are the most effective methods for confirming suspicious lesions or small liver cancers.

⑶ Magnetic resonance imaging (MRI): In liver cancer, T1 and T2 relaxation times are prolonged. In more than half of the cases, the tumor shows low signal intensity or equal signal intensity compared with the surrounding liver tissue on T1-weighted images, while high signal intensity is shown on T1-weighted images. The characteristics of primary liver cancer MRI are as follows: ① fatty degeneration of the tumor, short T1 relaxation time, equal or high signal on T1-weighted images, uneven high signal intensity on T2-weighted images, unclear lesion edges, and low signal intensity on liver cancer with fibrosis due to long T1 relaxation time. ② The presence of tumor capsule, T1-weighted images show a low signal intensity ring around the tumor, and T2-weighted images show unsatisfactory capsule. ③ The tumor invades the blood vessels. The advantage of MRI is that it can show the branches of the portal vein and hepatic veins and the compression and displacement of the blood vessels without the injection of contrast agents. In the case of tumor thrombus, the T1-weighted image shows medium signal intensity, and the T2-weighted image shows high signal intensity. ④ The sub-nodules have a higher signal intensity than normal liver parenchyma on T2-weighted images.

⑷ Angiography of primary liver cancer: Non-invasive methods such as ultrasound, CT, and MRI have been able to detect many small liver cancers. However, angiography still occupies a certain position in the diagnosis of liver cancer. Angiography can often make a more accurate and rapid diagnosis of small liver cancers with less than 2 views. At present, the Seleinger percutaneous stimulation of the femoral artery catheterization method is still used at home and abroad for liver angiography. The twisted catheter has the highest super-selective success rate. In order to diagnose liver cancer and understand the direction and anatomical relationship of the hepatic artery, the catheter can be inserted into the common hepatic artery or the proper hepatic artery to achieve the purpose. If vascular variation is suspected, selective superior mesenteric artery angiography can be added. If the purpose is embolization therapy, the catheter should be inserted as deep as possible to super-select the blood supply artery close to the tumor to reduce the impact on the blood supply to the non-tumor area. The angiography manifestations of liver cancer are: ① Tumor blood vessels and tumor staining are characteristic manifestations of small liver cancer. The arterial phase shows disordered tumor vascular proliferation, and the capillary phase shows tumor staining. Small liver cancer sometimes only shows tumor staining without vascular proliferation. After treatment, the reduction or disappearance of tumor blood vessels and changes in tumor staining are important indicators for judging the response to treatment. ② Larger tumors may show the following malignant features, such as straightening, twisting and displacement of arterial positions; tumor lakes, where contrast agents accumulate in the tumor during the arterial phase and are delayed in emptying; tumor encirclement of arteries, where tumor growth and infiltration cause the encircled arteries to be compressed irregularly or stiff; arteriovenous fistulas, where portal vein shadows are shown during the arterial phase; portal vein tumor thrombus formation, where a cord-like "velvet sign" parallel to the portal vein is seen in the venous phase, indicating that the portal vein has been invaded by the tumor. This sign can be seen during the arterial phase when an arteriovenous fistula is present. The ability of angiography to detect liver cancer depends on the number of new blood vessels in the lesion. Multivascular liver cancer is easy to show even if it is less than 2 cm or smaller. In recent years, digital subtraction angiography (DSA) has been developed, which uses an electronic computer to convert the video signal of the image into a digital signal, then amplifies the subtracted data signal and transfers it into a video signal, reconstructs the analog image output, and displays an angiography image with a clear background and enhanced contrast. The significance of hepatic angiography is not only in diagnosis and differential diagnosis, but also in estimating the scope of lesions before surgery or treatment, especially understanding the situation of sub-nodules spread in the liver; vascular anatomical variations and the anatomical relationship of important blood vessels and portal vein infiltration can provide correct and objective information. It is of great value for the possibility and thoroughness of surgical resection and for deciding a reasonable treatment plan. Angiography is not included in routine examination items and is only considered when the above non-invasive examinations are not satisfactory. In addition, angiography not only plays a diagnostic role, but some patients who are not suitable for surgery can immediately undergo chemotherapy embolization or the introduction of anticancer drugs or other biological immune preparations during angiography.

⑸ Radionuclide imaging Hepatobiliary radionuclide imaging uses gamma imaging or single photon emission computed tomography (SpECT). In recent years, efforts have been made to improve the imaging effect and to find radioactive drugs with high specificity and strong affinity. For example, radioimmunoassay diagnosis of anti-liver cancer monoclonal antibodies or related tumor markers labeled with radionuclides with strong specificity has begun to be used in clinical practice, which can effectively increase the cancer/liver ratio of radioactivity; 99mTc-pMT (99mTc-pyridoxal pentamethyltryptophan) is an ideal hepatobiliary imaging agent with a short hepatobiliary transit time. There is no bile duct system in liver cancer and hepatic adenoma for bile excretion and it has a certain affinity with pMT. Therefore, it can be concentrated and retained in liver cancer and hepatic adenoma for a long time. During delayed imaging (2 to 5 hours), 99mTc-pMT in liver cancer and hepatic adenoma tissues is still retained, while it has been emptied in the surrounding liver parenchymal cells, making the radioactivity in the cancer or adenoma much higher than that in normal liver tissue, resulting in a "hot zone". Therefore, it is clinically used for qualitative localization diagnosis of liver cancer, such as qualitative diagnosis of AFp-negative liver cancer, differentiation of primary and secondary liver cancer, diagnosis of extrahepatic metastasis and diagnosis of liver adenoma. Since the positive rate of hepatocellular carcinoma is only about 60% and affected by the resolution of the instrument, lesions within 2 cm are difficult to display, so its clinical application is not ideal.

3. Liver tissue biopsy or cytology In recent years, biopsy or fine needle aspiration under the guidance of real-time ultrasound or CT for histology or cytology is an effective method for confirming small liver cancer with a diameter of less than 2 cm. However, liver cancer near the edge is prone to liver cancer rupture, and there is also the risk of needle tract metastasis.

In summary, if AFp is significantly elevated, combined with typical superconducting images, primary liver cancer can be preliminarily diagnosed; for those with negative or low AFp concentrations, liver cancer markers other than AFp should be appropriately selected. Image diagnosis also has qualitative and positional diagnostic value. CT examination with contrast agent enhancement or dynamic enhancement scanning is helpful for liver cancer diagnosis. The characteristic manifestations of magnetic resonance imaging can assist in the diagnosis and differential diagnosis of liver cancer, and radioimmunoassay diagnosis has a high specificity. The diagnosis of microhepatic cancer and its differentiation from certain small benign lesions still need to be further studied.

Laboratory examination indicators of liver cancer mainly include liver cancer markers, liver function, hepatitis virus markers, immune function indicators, etc.

1. Liver cancer markers An ideal liver cancer marker should be present in the patient's serum, have high sensitivity and specificity, and accurately reflect its load level, so that it can be used for auxiliary diagnosis, efficacy evaluation, prognosis determination, and recurrence. ① Alpha-fetoprotein (AFp): As we all know, AFp is the most specific marker for diagnosing liver cancer, but there are still 30% to 40% false negatives and 2% false positives. At present, there are dozens of liver cancer markers reported in the literature, mainly the following categories: AFp, AFp monoclonal antibody, and AFp heterogeneity. ②Serum enzymes: γ-glutamyl transpeptidase (GGT) and its isoenzymes, fucosidase (AFU), α1-antitrypsin (AAT), aldolase isoenzyme A (ALD-A), alkaline phosphatase isoenzyme Ⅰ (ALp-Ⅰ), 5'-nucleoside phosphodiesterase isoenzyme V (5-Npp-Ⅴ), pyruvate kinase isoenzyme (pyK), placental glycosylation-transferase (GST), etc. ③Other markers such as abnormal prothrombin (DCp), ferritin and acid ferritin, etc. While searching for new and more effective specific liver cancer markers, people also optimize the screening, optimal combination and comprehensive observation of the various existing liver cancer markers, which greatly improves the accuracy of liver cancer diagnosis.

(1) AFp markers:

①AFp has become the best marker for liver cancer diagnosis: Since AFp was discovered in 1956, it was confirmed in 1964 that AFp can be detected in the serum of liver cancer patients, and it was widely used in clinical practice in the late 1960s. AFp has become the best marker for liver cancer diagnosis. AFp was first discovered by Bergstrand and Czar in human fetal serum in 1956. It is an embryo-specific alpha globulin synthesized by embryonic liver parenchymal cells and yolk sac cells. AFp begins to appear in the blood of the fetus from 6 weeks, reaching its peak at 12 weeks, with a content of more than 4g/L. AFp in serum can no longer be detected by conventional methods 5 weeks after birth. Radioimmunoassay determination of normal people is 1 to 20µg/L. In 1963, Abelev first discovered that mice inoculated with liver cancer can synthesize AFp, and then Tatarinov detected AFp in the serum of patients with primary liver cancer, and thus it was widely used in clinical practice and surveys. In addition, a few cases of pregnancy, active liver disease, genital embryonic tumors, secondary liver cancer and digestive tract cancer may also be positive for serum AFp. In 1977, the 1st National Liver Cancer Collaborative Conference proposed the standard for single AFp detection for diagnosis of primary liver cancer: AFp convection method positive or quantitative ≥400µg/L, lasting for more than 2 months, and excluding pregnancy, active liver disease, and genital embryonic tumors. Since AFp detection has been used in natural population and high-risk population surveys, a large number of studies have shown that AFp can increase the detection rate of subclinical liver cancer, thereby significantly improving the opportunity for early diagnosis and early treatment of liver cancer patients, and the five-year survival rate has increased several times, becoming the most specific tumor marker for detecting liver cancer. AFp is a glycoprotein composed of 590 amino acids, with sugar accounting for 4%, a molecular weight of 64,000 to 700,000, an isoelectric point of 4.75, a sedimentation coefficient of 4.55, and a half-life of 3 to 7 days. There is heterogeneity in the sugar content and sugar chain structure of AFp from different tissues. AFp with roughly the same molecular structure but different sugar chains or protein isoelectric points are called AFp variants. AFp variants can be separated by using the difference in the binding ability of exogenous plant lectins and AFp sugar chains.

② AFp detection methods: agar diffusion method, countercurrent immunoelectrophoresis method, hemagglutination method, radioimmunoassay, rocket electrophoresis autoradiography, enzyme-linked immunosorbent assay, etc. Among them, the diffusion method and the countercurrent method are less used because they are not sensitive enough.

③The clinical application value of AFp:

A. AFp is a highly specific indicator for the clinical diagnosis of primary liver cancer: clinically, about 60% to 70% of primary liver cancers have elevated AFp. If diagnosed according to the standard, the false positive rate is only 2%.

B. Differential diagnosis of primary liver cancer and other liver diseases: AFp in the serum of patients with primary liver cancer often persists above 500µg/L, and ALT is mostly normal or slightly elevated. Or AFp is positive at a low concentration, but it often rises continuously or remains unchanged or changes in a saddle shape, and is inconsistent with the dynamic changes of ALT. However, the serum AFp content of patients with chronic active liver disease is rarely above 400µg/L, and the increase in AFp is often transient, not exceeding 2 months, and is parallel to the changes in ALT. Detection of AFp isomers is more conducive to identification. Lentil agglutinin (LCA)-bound AFp only accounts for 3% to 6.3% of the total AFp in patients with chronic hepatitis and cirrhosis, but accounts for 45% to 47.8% in primary liver cancer. Taking the percentage of LCA-bound AFp ≥25% as the diagnostic standard for liver cancer, the sensitivity is 73.5% to 84.3% and the specificity is 97% to 98.6%.

C. Improve the early diagnosis rate: It can be diagnosed 6 to 12 months before the clinical symptoms of liver cancer appear, and liver cancer can be found early through census. In Shanghai from 1971 to 1976, a census of 1.96 million people was conducted and 300 cases were detected, of which 134 were subclinical liver cancer, accounting for 44.4%.

D. Evaluate the efficacy of surgery or other treatments and judge the prognosis: For AFp-positive liver cancer that has undergone radical resection, AFp will turn negative within 1 to 2 months after surgery. If AFp cannot be reduced to normal after surgery or rises again after it has been reduced, it indicates that cancer cells are still present. It is a sensitive indicator that reflects the dynamic changes of the disease and evaluates the efficacy. Observing the changes in AFp after other treatments for liver cancer patients can also be used to judge the efficacy and estimate the prognosis.

E. Early detection of postoperative recurrence and metastasis: Monthly follow-up with AFp and B-ultrasound after surgery can detect recurrent cancer early for timely treatment. The interval between examinations can be relatively extended after two years. Subclinical recurrence and metastasis after radical resection can be detected.

④ Diagnostic criteria for liver cancer: The diagnostic criteria for liver cancer established in 1977 are that if there is no evidence of other liver disease activity, pregnancy and gonadal embryonal tumors are excluded, liver cancer can be diagnosed if AFp ≥ 500 µg/L lasts for 1 month or AFp ≥ 200 µg/L lasts for 2 months. In recent years, the diagnostic criteria for liver cancer have been supplemented in combination with imaging examinations. In 1990, the "Chinese Common Malignant Tumor Diagnosis and Treatment Standards for Primary Liver Cancer" compiled by the National Cancer Prevention and Control Office and the Chinese Anti-Cancer Association added the following:

A. If there is no other evidence of liver disease, AFp>400µg/L by convection or radioimmunoassay for more than 4 weeks, and pregnancy, active liver disease, gonadal embryonal tumors, and metastatic liver cancer can be excluded.

B. Patients with obvious liver parenchymal space-occupying lesions on imaging examinations, who can exclude hepatic hemangioma and metastatic liver cancer, and who meet one of the following conditions: a. AFp ≥ 200 µg/L; b. Typical imaging manifestations of primary liver cancer; c. No jaundice but significantly increased ALp or GGT; d. Clear metastatic lesions or bloody ascites at a distance, or cancer cells found in the ascites; e. Clear cirrhosis with positive hepatitis B markers.

⑤ Increased AFp in non-cancerous liver diseases: In addition to primary liver cancer, AFp often increases transiently in non-cancerous liver diseases, especially viral hepatitis and cirrhosis (see Table 2). Research data show that in non-cancerous liver diseases, AFp concentrations are mostly below 200µg/L and are transient. The mechanism of increased AFp is related to the regeneration of liver cells. In addition, the de-inhibition of AFp synthesis genes by hepatitis viruses may also be one of the mechanisms of increased AFp. Since most liver cancers occur on the basis of chronic liver disease, patients with liver diseases with high AFp levels should be alert to the possibility of combined liver cancer. From the above data, AFp in non-cancerous liver diseases is mostly below 200µg/L and is mostly transient, while liver cancer is mostly above 200µg/L, showing a continuous peak or dynamic rise. Therefore, dynamic observation of AFp concentration changes combined with imaging examinations can basically obtain a correct diagnosis. In other diseases such as embryonal carcinoma, gastric cancer and other tumors, AFp is sometimes significantly increased, which causes certain difficulties in clinical diagnosis. In a small number of digestive tract cancers, especially gastric cancer with secondary liver metastasis, about 15% of patients are AFp positive, but the vast majority of AFp levels are below 100µg/L, and only 1% to 2% of patients may be higher than 200µg/L, with the highest reaching more than 120,000µg/L. If the primary liver cancer lesion is removed and the metastatic lesion is retained, AFp can be reduced to normal levels.

(2) AFp heterogeneity: AFp is a group of glycoproteins. The sugar chain structures of AFp that is elevated in primary liver cancer, secondary liver cancer, embryonic cell tumors and various benign liver diseases are different. AFp molecules bound by exogenous lectins can form molecular heterogeneities with various heterogeneities. Some heterogeneous AFp have affinity for concanavalin A (ConA), while others have affinity for lentil lectin (LCA). Therefore, AFp is divided into ConA-affinity and LCA-affinity types, and LCA-affinity and LCA-infinity types. Heterogeneity analysis can be used to identify various AFp-elevated diseases. Data show that different heterogeneity contents can be used to identify benign and malignant liver diseases, and the positive detection rate for liver cancer is as high as 85% or more. However, it should be noted that benign liver diseases have about 30% false positives. In studies of liver cancer and embryonic tumors, it was found that liver cancer AFp is mainly ConA-affinity, while teratomas and gastric cancer liver metastases are mainly ConA-inaffinity.

(3) Abnormal prothrombin (DCp): Prothrombin precursor cannot be converted into prothrombin and released into the blood after lack of vitamin K or taking vitamin K antagonists, which is abnormal prothrombin (Ap). The glutamic acid residue at its N-terminus is not carboxylated, so it is also called decarboxy prothrombin (DCp). It is inactive in general coagulation tests. Abnormal prothrombin does not exist in normal human plasma. In 1984, it was discovered that DCp can be measured in liver cancer patients and may be a marker for liver cancer. The increase in DCp in the serum of patients with primary liver cancer is different from the increase in DCp caused by vitamin K deficiency due to biliary obstruction or other reasons. The two can be distinguished through vitamin K treatment tests. Compared with AFp, DCp, as a single liver cancer marker, has a positive rate of 67.3%, but the false positive rate of abnormal prothrombin caused by benign liver disease is lower than that of AFp, so it is better than AFp in distinguishing benign liver disease. The combination of the two can reduce false positives, so it can be used to distinguish benign and malignant liver diseases. In AFp-negative liver cancer, the DCp positive rate remains high, up to 61.9%, so it helps in the diagnosis of AFp-negative liver cancer. The serum DCp positive rate of patients with small liver cancer is only 19%, the positive rate of patients with liver cancer with a diameter of 3 to 5 cm is about 55.6%, and the positive rate of large liver cancer is 66.2%, while the DCp positive rate of chronic liver disease is only 14.8%. Therefore, although DCp is helpful for the diagnosis and differential diagnosis of liver cancer, it is not ideal for the early diagnosis of liver cancer.

(4) α-L-fucosidase (AFU): AFU is a lysosomal hydrolase widely present in human and animal tissue fluids, and is involved in the degradation and metabolism of various biologically active substances such as glycoproteins and glycolipids. Its activity can be detected by spectrophotometric colorimetry or fluorescence colorimetry, and the normal value is 450mmol/(ml·h). The AFU activity in the serum of patients with hepatocellular carcinoma is significantly higher than that of patients with cirrhosis and secondary liver cancer. The sensitivity for the diagnosis of primary liver cancer is 75%, the specificity is 90%, and the positive detection rate for AFp-negative liver cancer is 80.8%. However, high AFU can also be seen in viral hepatitis, diabetes, exophthalmos goiter, and gastrointestinal cancer.

(5) Alkaline phosphatase (ALp) isoenzymes: ALp can be elevated in the serum of patients with various obstructive jaundice. 80% to 90% of patients with liver cancer have elevated ALp, and about 10% of patients with small liver cancer have positive ALp, but it lacks specificity. ALp isoenzyme I (ALp-I) is relatively specific for the diagnosis of liver cancer, but the positive rate is low, only 24.8%. Due to its low sensitivity, it is not suitable as a screening indicator for liver cancer. Since both primary and metastatic liver cancer can cause obstructive jaundice, the ALp concentration is proportional to the degree and duration of obstruction. Therefore, a comprehensive analysis should be made in differential diagnosis.

(6) 5'-nucleotide phosphodiesterase isozyme V (5'-NpDV): 5'-NpDV can be found in the serum of patients with liver cancer. The positive rate is 84.6% to 85.7% in AFp-positive liver cancer and 56.4% to 91.0% in AFp-negative liver cancer. However, its specificity is not high. It can be found in both benign liver lesions and metastatic liver cancer, especially in the latter, with a positive rate of up to 83%.

(7) GGT isoenzymes: The GGT positive rate in patients with liver cancer is as high as over 90%, but lacks specificity. Among them, there are three tumor-related isoenzymes (Ⅰ', Ⅱ', Ⅲ'), collectively referred to as GGT-Ⅱ, with a positive rate of 79% in patients with liver cancer, 84.0% in AFp-negative liver cancer and 78.6% in small liver cancer. In recent years, the quantitative determination of GGT-Ⅱ has improved the positive diagnosis rate to a certain extent. It is currently believed that GGT-Ⅱ still has great diagnostic value for liver cancer.

(8) Ferritin and iso-ferritin: Ferritin can be divided into three types, A, B, and C, by isoelectric focusing and polyacrylamide gel electrophoresis, which are alkaline, neutral, and acidic ferritin, respectively. Total ferritin is not highly specific for liver cancer diagnosis, while acidic ferritin is mostly produced by liver cancer cells and has a relatively high specificity. The positive rate in liver cancer patients is about 80%.

(9) Aldolase (ALD) isozyme: ALD is one of the key enzymes for fructose decomposition during glycolysis. Type A comes from muscle, type B comes from liver, and type C comes from brain tissue. Type A is the main type in the fetal period. ALD-A is elevated in primary liver cancer, with a positive rate of 76%. It is also elevated in metastatic liver cancer and other digestive tract malignancies, such as adenocarcinoma. The positive rate of ALD-A is not high and lacks specificity.

(10) Pyruvate kinase isoenzyme (M2-pyK): Pyruvate kinase (pyK) is a key enzyme in the glycolysis process. It has four isoenzymes: L, R, M1 and M2. The M2-pyK positivity rate in patients with hepatocellular carcinoma can reach 93%, while that in patients with benign liver diseases is mostly normal. Elevated M2-pyK levels can also be seen in other gastrointestinal malignancies, muscle diseases and hemolytic diseases.

(11) Glutathione S-transferase (GST): The serum GST level of liver cancer patients is 25 times that of normal people, and the positive rate of liver cancer diagnosis is 89.5%. However, it is lower in patients with chronic liver disease, which helps to distinguish liver cancer from benign liver disease.

(12) αl-antitrypsin (αl-AT): αl-AT is a protease inhibitor synthesized by hepatocytes. The diagnostic positive rate for primary liver cancer can reach 86.7%, but the specificity is only 50%. It can be increased in inflammatory bowel disease and benign liver disease, and the false positive rate is high.

Other markers: Other liver cancer markers include α1-AAT, α1-AAT isoforms, pseudouridine, CA19-9, CA50, etc. They have high sensitivity and specificity for liver cancer, but cannot effectively and specifically distinguish primary and secondary liver cancer. In recent years, it has been reported that serum type IV collagen has differential diagnostic value in benign and malignant liver tumors, with an accuracy rate of 90.8%. The sensitivity and specificity for liver cancer are 91.48% and 89.65% respectively. When combined with AFp, the sensitivity can reach 95.74%.

(13) Recent studies on AFpmRNA have revealed that some substances closely related to metastasis can be detected in the serum of liver cancer patients. These substances can be used as indicators to judge whether liver cancer has metastasized, the progression of the disease, and the prognosis.

The positive expression rate of AFp mRNA in liver cancer tissue is 76.9%. In liver cancer tissues with chronic liver disease, especially moderate and severe liver cirrhosis, the positive expression rate of AFp mRNA can reach 88.8%, and that of adjacent tissues is 69.4%. The positive expression rate of AFp mRNA in liver cancer tissues without chronic liver disease or liver cirrhosis is 50%, and there is no AFp expression in adjacent tissues. It can be seen that AFp protein is specifically expressed by liver cancer cells. In 1994, Matsumura of Japan used nested reverse transcription polymerase chain reaction (RT-PCR) to detect the nucleated cell components of peripheral blood of 33 liver cancer patients and found that 17 cases (52%) had AFp mRNA. AFp mRNA expression was found in the serum of 15% of patients with liver cirrhosis and 12% of patients with chronic hepatitis, but no expression was found in the serum of normal people. The overexpression of AFp mRNA in liver cancer patients has no obvious relationship with HBV markers, but is related to tumor size and serum AFp levels. The positive expression rate of AFpmRNA in the serum of patients with metastatic liver cancer was 100%, while that of patients without metastasis was 41%. The positive expression of AFpmRNA indicates the presence of scattered liver cancer cells in the peripheral blood. Since the metastasis of tumors depends on the balance between the selectivity of tumor cells and the reactivity of the host, only tumor cells may appear in the blood circulation without the formation of metastatic foci. However, AFpmRNA can be used as an indicator of whether liver cancer has micrometastatic foci, which has important reference value for the prognosis of liver cancer and the selection of liver transplant recipients. The relationship between AFpmRNA and liver cancer stage, portal vein tumor thrombus, satellite nodules, tumor size, serum AFp concentration and distant metastasis was analyzed, and it was found that the serum AFpmRNA positive rate of patients with stage III liver cancer reached 73.3%, while that of stage I was 19.35%. For patients with intrahepatic satellite cancer nodules, portal vein cancer thrombus, cancer lesions larger than 5 cm in diameter, serum AFp greater than 400 µg/L, and distant metastasis, the positive rates were 73.33%, 91.67%, 47.37%, 50.91%, and 100%, respectively, which were significantly higher than the average level (44.87%). This indicates that with the malignant evolution of liver cancer, the positive detection rate of AFpmRNA in serum gradually increases, which can reflect the trend of postoperative liver cancer recurrence or metastasis to a certain extent, and can be used as one of the objective indicators for clinical judgment of liver cancer prognosis.

(14) CD44vmRNACD44v is an adhesion molecule present on the cell surface, involved in cell-cell and cell-stroma reactions. CD44v refers to the standard CD molecule that is spliced ​​with at least 5 exons in different combinations. A single cell can express 2 or more CD44v, which can enable tumor cells to adhere to the host cell extracellular matrix or basement membrane, promoting cancer cell invasion and metastasis.

There are many kinds of CD44v in liver cancer cells. By specifically combining and amplifying tumor CD44v primers and detecting CD44vmRNA in peripheral serum, it is possible to determine whether there are liver cancer cells in the peripheral blood and the invasive ability of these cancer cells. Therefore, CD44vmRNA can also be used as a marker to determine the prognosis and metastasis of liver cancer.

Liu Pengfei et al. reported that CD44vmRNA can be detected in 66.7% (10/15 cases) of liver cancer serum. After surgical resection of the tumor, the recurrence rate of CD44vmRNA positive group reached 100%, while that of negative group was only 25%. In AFp negative liver cancer patients, CD44v is still positively expressed, suggesting that CD44v can be used as an important complementary indicator of AFp in the diagnosis of liver cancer and monitoring of postoperative recurrence.

(15) Intercellular adhesion molecule-I: Recently, some scholars have found that a substance on the surface of liver cancer cells, intercellular adhesion molecule-I, is stably expressed in liver cancer cells. When liver cancer cells metastasize, its expression level can increase exponentially. Therefore, intercellular adhesion molecule-I is expected to become a new marker for liver cancer metastasis. Since liver cells can synthesize albumin, the detection of peripheral blood albumin mRNA can also be used as a reference indicator for predicting liver cancer metastasis.

2. Liver function test Liver function test is of great reference significance for understanding the background information of liver disease, the current status of liver function, guiding liver cancer treatment and judging prognosis.

In the early stages of liver cancer, bilirubin is generally within the normal range and clinically there is no jaundice. A few tumors located in special parts such as the liver hilum may cause obstructive jaundice due to compression of the bile duct. Bilirubin is progressively elevated, which often indicates that the disease is in the late stage, mainly due to compression by enlarged cancer masses or severe liver function damage. Albumin/globulin inversion is an important indicator of liver decompensation. Since liver cancer is often combined with chronic liver damage such as cirrhosis, it often appears in the early stage and becomes more significant as the size of liver cancer increases and liver function damage worsens. Serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) is released into the blood due to liver cell damage. The transaminase level is an indicator of the degree of liver cell damage. Due to liver function damage, reduced coagulation factor production, and prolonged prothrombin time, when it is extended to more than twice the normal value, it indicates that the patient is difficult to tolerate surgery. A significant increase in GGT is often due to a huge tumor, portal vein cancer thrombosis, or combined active hepatitis. Patients with excessively high GGT have a poor prognosis.

3. Hepatitis virus serology Hepatitis virus, especially hepatitis B virus infection, is closely related to liver cancer in my country. HBV serological markers are an important reference for diagnosing liver cancer. The HBsAg positive rate of liver cancer patients is 89.5% (12.5% ​​in the normal population), and anti-HBcAb is 96.5%. If the five serological indicators of HBV (HBsAg, HB-sAb, HBeAg, HBeAb, HBeAb) are all negative, and HBV-DNA is also negative, the possibility of liver cancer is very small, which can be used to distinguish it from secondary liver cancer.

4. Immunological indicators Immunological indicators are tests of the body's immune function, which help to understand the body's disease resistance and treatment effects. Commonly used indicators include tuberculin test (OT test), lymphocyte transformation test, natural killer cells (NK), macrophage activity, etc.

5. Trace elements Through the detection and analysis of trace elements in the serum of patients with hepatitis, cirrhosis, liver cancer and normal people, it was found that the serum Cu content in patients with liver cancer was significantly higher than that in the cirrhosis group and the normal control group. The serum Cu level was not related to the AFp level, but had a certain relationship with the patient's prognosis. It was significantly increased in the liver cancer and cirrhosis groups, and the serum Al, serum Mg and blood selenium contents were significantly reduced.

6. Liver puncture biopsy Liver puncture biopsy is helpful in confirming the diagnosis. However, due to its low positive rate, it may cause bleeding, tumor rupture and needle tract metastasis, etc., and is generally not used as a routine method. For small liver lesions that cannot be diagnosed, fine needle aspiration biopsy under B-ultrasound is expected to obtain pathological evidence.