Application of methylation level and protein concentration combined detection in HBV-HCC early warning

Abstract

The invention provides an application of joint detection of methylation level and protein concentration in early warning of hepatitis B virus related hepatocellular carcinoma, which adopts joint detection of methylation level of serum TERT gene promoter and TERT protein concentration and is applied to early warning of hepatitis B virus related hepatocellular carcinoma. According to the invention, a method for detecting the methylation level of a TERT gene promoter in free DNA of serum and a method for detecting the protein concentration of TERT in serum are utilized, and the two detection methods are combined into a scheme as a clinical prediction target, so that early warning of hepatitis B virus related hepatocellular carcinoma is realized.

Description

Technical Field

[0001] This invention relates to the field of early warning technology for hepatitis B-related hepatocellular carcinoma, specifically to the application of a combined detection of methylation level and protein concentration in early warning of HBV-HCC. Background Technology

[0002] Primary hepatocellular carcinoma (HCC) is a group of malignant tumors with high incidence and mortality rates. Its main causes include hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, long-term heavy alcohol consumption, metabolic-related fatty liver disease, and aflatoxin exposure. Hepatocellular carcinoma (HCC) is the main pathological type of primary liver cancer. In China, HBV infection is the most significant causative factor of HCC, with approximately 80% of HCC cases being hepatitis B virus-associated hepatocellular carcinoma (HBV-HCC). In this invention, HBV-HCC will be used to refer to hepatitis B-related liver cancer. The large number of people with chronic HBV infection is a major reason for the high incidence of HBV-HCC in China.

[0003] Despite significant advancements in HBV-HCC diagnosis and treatment technologies in recent years, the 5-year overall survival rate remains low, at only 14.1%. The primary reason is the lack of early warning biomarkers with both high sensitivity and specificity, and reliable early screening protocols for high-risk populations. This results in 70% to 80% of patients being diagnosed at an advanced stage. Commonly used biomarkers such as alpha-fetoprotein (AFP), alpha-fetoprotein-L3 (AFP-L3), and abnormal prothrombin (DCP) all have limitations and cannot meet the early warning needs of high-risk populations. Therefore, developing reliable early warning protocols is of paramount importance. Summary of the Invention

[0004] The purpose of this invention is to propose an application of combined detection of methylation level and protein concentration in the early warning of HBV-HCC. The invention uses the combined detection of serum TERT gene promoter methylation level and TERT protein concentration for the early warning of HBV-HCC.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides an application of combined detection of methylation level and protein concentration in early warning of HBV-HCC. It uses the combined detection of serum TERT gene promoter methylation level and TERT protein concentration for early warning of hepatitis B virus-associated hepatocellular carcinoma (HBV-HCC).

[0007] Furthermore, the combined detection of TERT gene promoter methylation level and TERT protein concentration specifically involves combining two detection methods—detection of TERT gene promoter methylation level in serum cell-free DNA and detection of serum TERT protein concentration—into a single scheme as a clinical prediction target, thereby achieving early warning of HBV-HCC.

[0008] Furthermore, the detection of TERT gene promoter methylation level in serum cell-free DNA includes the following steps:

[0009] (1) Collect serum and perform plasma separation;

[0010] (2) Extraction of cell-free DNA;

[0011] (3) Detection of methylation level.

[0012] Furthermore, the plasma separation employs a two-step centrifugation method:

[0013] The serum was centrifuged at a low speed, and the first supernatant was collected into a low-adsorption tube.

[0014] Then, the first supernatant is centrifuged at a high speed, and the second supernatant is collected. The second supernatant is then transferred, aliquoted, and stored to avoid aspirating white blood cells or red blood cells.

[0015] Furthermore, the cell-free DNA extraction includes the following steps:

[0016] a. Add protease lysis buffer to dissolve the protease. Add the dissolved protease, magnetic beads and plasma sample to a centrifuge tube, mix well, add lysis buffer, mix well and vortex.

[0017] b. After centrifugation and sedimentation, place the centrifuge tube on a magnetic rack for adsorption and discard the third supernatant;

[0018] c. Add the first and second washing solutions sequentially, shaking to mix well after each addition, then adsorb and discard the fourth supernatant.

[0019] d. Repeat the second washing solution, shake to mix, adsorb, discard the residual liquid, and air dry at room temperature;

[0020] e. Add the elution buffer, vortex to mix, and then vortex again.

[0021] f. After placing the centrifuge tube on a magnetic rack for adsorption, collect the supernatant, i.e., the free DNA solution, and store it below -10°C.

[0022] Furthermore, the methylation level detection is performed using amplicon methylation sequencing (MultiplexBS) combined with bisulfite treatment and multiplex PCR amplification, specifically including the following steps:

[0023] ① DNA sample testing;

[0024] ② Construction of amplicon methylation library;

[0025] ③ High-throughput sequencing and data processing.

[0026] Furthermore, step ① DNA sample detection includes:

[0027] a. Use agarose gel electrophoresis to detect the degree of DNA degradation and contaminant bands;

[0028] b. Use Qubit 3.0 to quantify DNA concentration, ensuring that the DNA concentration is ≥5ng / μL and the total amount is ≥50ng for library construction.

[0029] Furthermore, step ②, the construction of the amplicon methylation library, includes:

[0030] Design of TERT gene promoter region-specific primer sequences;

[0031] Sulfite treatment of free DNA;

[0032] PCR amplification using specific primers;

[0033] PCR amplification using index primers.

[0034] Furthermore, step ③, high-throughput sequencing and data processing, includes:

[0035] a. After the library quality is qualified, high-throughput sequencing is performed using the Illumina platform;

[0036] b. Filter the sequencing data to remove adapters and low-quality sequences, and use BSMAP software to align with the reference genome;

[0037] c. By analyzing the variation patterns of CpG sites, the methylation status of the target gene region can be accurately assessed.

[0038] d. Furthermore, the serum TERT protein concentration was determined using a kit that employed a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA).

[0039] The specific steps are as follows:

[0040] a. Add human telomerase reverse transcriptase (TERT) calibrator and the sample to be tested to a microplate pre-coated with anti-human telomerase reverse transcriptase (TERT) antibody;

[0041] b. Add biotin-labeled anti-human telomerase reverse transcriptase antibody, mix well and incubate;

[0042] c. After incubation, wash, add HRP-conjugated avidin, mix well, and incubate again;

[0043] d. After incubation, wash to remove unbound components and form a sandwich complex of solid-phase antibody-antigen-biotin-labeled antibody-avidinase on the solid-phase surface;

[0044] e. Add the chromogenic substrate (TMB chromogenic solution), the reaction produces a blue product, which turns yellow after the reaction is terminated;

[0045] f. Measure the absorbance (OD value) at a wavelength of 450 nm using an ELISA reader. The absorbance is positively correlated with the TERT concentration in the sample.

[0046] g. Calculate the concentration of TERT protein in the sample based on the calibration curve. The kit's detection range is 0.312 ng / ml - 10 ng / ml.

[0047] The present invention has the following beneficial effects:

[0048] 1. This invention provides an application of combined detection of methylation level and protein concentration in early warning of HBV-HCC, which uses combined detection of serum TERT gene promoter methylation level and TERT protein concentration for early warning of HBV-HCC.

[0049] 2. This invention utilizes a method for detecting the methylation level of the TERT gene promoter in cell-free DNA and a method for detecting the concentration of TERT protein in serum, combining these two detection methods into a single scheme as a clinical prediction target, thereby achieving early warning of HBV-HCC.

[0050] 3. In this embodiment of the invention, the amplicon methylation sequencing protocol provides accurate and reliable results by measuring the methylation level of the TERT gene promoter in serum cf DNA; while the ultrasensitive ELISA method for detecting serum TERT protein concentration is simple to operate and cost-effective. Both detection methods require only a small amount of peripheral serum sample to complete the analysis, demonstrating high practicality and convenience. The combined detection protocol provided by this invention has broad clinical application potential and can be used for early warning of high-risk groups for HBV-HCC. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0052] Figure 1The results of a clinical trial on the detection of TERT gene promoter methylation level in serum cf DNA in the application of combined detection of methylation level and protein concentration in early warning of HBV-HCC provided by an embodiment of the present invention.

[0053] Figure 2 The results of serum TERT protein concentration detection in a clinical trial 2 provided by an embodiment of the present invention for the application of combined detection of methylation level and protein concentration in early warning of HBV-HCC;

[0054] Figure 3 This invention provides an application of combined methylation level and protein concentration detection in early warning of HBV-HCC, including ROC curve analysis of HBV-HCC patients. Detailed Implementation

[0055] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] Human TERT protein is the catalytic subunit of telomerase. While its expression is low in normal cells, it is typically highly active in most malignant tumors, especially HBV-HCC. In more than half of chronic HBV-infected individuals, integration of the HBV gene with the TERT gene is detectable. This integration event leads to increased methylation levels in the TERT gene promoter region, thereby promoting TERT protein overexpression. These changes may make this population a high-risk group for HBV-HCC. Therefore, detecting TERT gene promoter methylation levels and quantitatively measuring TERT protein concentration in chronic HBV-infected patients at different disease stages could help patients understand disease progression early and provide support for early warning of HBV-HCC.

[0057] This invention provides an application of combined detection of methylation level and protein concentration in the early warning of HBV-HCC. By measuring the methylation level of the telomerase reverse transcriptase (TERT) gene promoter in peripheral blood cell-free DNA (cf DNA) and the concentration of TERT protein in peripheral blood, it is used for the early warning of hepatitis B virus-related hepatocellular carcinoma (HBV-HCC). Specifically, a combined detection scheme is used to screen high-risk groups who may develop HBV-HCC in the early stage.

[0058] This invention provides an application of combined methylation level and protein concentration detection in early warning of HBV-HCC. It utilizes a method for detecting the methylation level of the TERT gene promoter in serum free DNA and a method for detecting serum TERT protein concentration, combining these two detection methods into a single scheme as a clinical prediction target, thereby achieving early warning of HBV-HCC.

[0059] In this embodiment of the invention, the method for detecting the methylation level of the TERT gene promoter in cell-free DNA (cf DNA) includes the following steps:

[0060] (1) Collect serum and then perform plasma separation:

[0061] a. Collect 5ml of serum using an EDTA anticoagulant blood collection tube, gently invert and mix 4 times, then place at 4℃ and complete plasma separation within 8 hours; or collect serum using a room temperature stable tube, store at room temperature, and complete plasma separation within 96 hours to avoid hemolysis.

[0062] b. Plasma separation uses a two-step centrifugation method: First, centrifuge the serum at 4℃ and 1600g for 10 minutes (for serum samples transported at room temperature, centrifugation for 15 minutes is required). The resulting supernatant is the first supernatant, which is collected into a low-absorption tube. Then, centrifuge the serum at 4℃ and 16000g for 10 minutes. The resulting supernatant is the second supernatant, which is transferred and aliquoted and stored at -20℃ or -80℃. Avoid aspirating white blood cells or red blood cells.

[0063] (2) Extraction of cell-free DNA from plasma:

[0064] a. Add proteinase K dissolving solution according to the indicated amount on the dry powder proteinase K reagent bottle, and gently invert to fully dissolve the proteinase K; add 20 μL of dissolved proteinase K, 30 μL of magnetic beads G and 400 μL of plasma sample to a 2 mL centrifuge tube, vortex to mix, then add 500 μL of lysis buffer MLK, mix well, and incubate at 40°C with shaking for 10 minutes (or let stand at room temperature for 15 minutes, vortexing intermittently).

[0065] b. After centrifugation and brief settling, place the centrifuge tube on a magnetic rack for 3 minutes to attract the supernatant;

[0066] c. Add 500 μL of the first washing solution (i.e., washing solution 1) and the second washing solution (i.e., washing solution 2) in sequence, shake to mix well after each addition, adsorb, and discard the supernatant.

[0067] d. Repeat the addition of the second washing solution once, shake to mix well, adsorb, and finally discard the residual liquid (i.e., the supernatant), and air dry at room temperature for 5 minutes;

[0068] e. Add 43 μL of elution buffer AE, vortex to mix, and then place at 40°C and vortex for 5 minutes.

[0069] f. Place the centrifuge tube on a magnetic rack. After adsorption, collect the supernatant, i.e., the cf DNA solution. Store the cf DNA solution at -30℃ to -10℃ for short-term storage and below -70℃ for long-term storage. In this technical field, it is generally considered that short-term storage is within 2 years and long-term storage can reach more than 5 years.

[0070] It should be noted that the magnetic beads mentioned above are small beads with magnetic adsorption properties included in the kit, which can play an adsorption role. The first washing solution and the second washing solution are washing solution 1 and washing solution 2, respectively.

[0071] (3) Methylation level detection:

[0072] The amplicon methylation sequencing (MultiplexBS) technique, combined with bisulfite treatment and multiplex PCR amplification, specifically includes the following steps:

[0073] ① DNA sample detection: Agarose gel electrophoresis was used to detect the degree of DNA degradation and impurities. The DNA concentration was quantified using a Qubit 3.0 analyzer, ensuring a DNA concentration ≥5 ng / μL and a total amount ≥50 ng for library preparation.

[0074] ② Construction of amplicon methylated libraries: Methylated libraries were constructed using bisulfite treatment and multiplex PCR amplification techniques. The library concentration was initially quantified using Qubit 3.0, and the insert size was detected using Caliper. The effective concentration of the library (3 nM) was accurately determined using qPCR to ensure library quality.

[0075] The construction of the amplicon methylation library mentioned above (②) specifically includes the following steps:

[0076] ②-1 Design of TERT gene promoter region-specific primer sequences

[0077] Because it is necessary to amplify short DNA fragments from cell-free plasma (cf DNA), the length of the amplified product is set to 100-160 bp to ensure reliability. The TERT gene promoter region is located on human chromosome 5 (chr5).

[0078] chr5 TERT: 5' 1294662 - 1297162 3'

[0079] The specific primer sequences are as follows:

[0080]

[0081] ②-2 Sulfite treatment of free DNA

[0082] ②-2.1 Sample preparation: Take 40 μl of DNA sample (add water if the volume is insufficient), add 110 μl of bisulfite conversion solution, mix well and centrifuge briefly.

[0083] ②-2.2 Thermal cycling treatment: Run the program in the PCR instrument (a. High temperature pretreatment: set the temperature to 98℃ and run the program for 10 minutes; b. Isothermal reaction: set the temperature to 64℃ and run the program for 2.5 hours; c. Low temperature storage: set the temperature to 4℃ and store for 20 hours).

[0084] ②-2.3 Purification and Adsorption:

[0085] a. Add the reaction solution to the adsorption column containing 600 μl of binding solution, invert to mix, and centrifuge (set the centrifuge relative centrifugal force ≥10000×g, centrifuge for 1 minute), and discard the filtrate.

[0086] b. Add 100 μl of washing solution, centrifuge, and discard the filtrate; add 200 μl of desulfonate solution, let stand at room temperature for 15 minutes, and then centrifuge.

[0087] c. Repeat centrifugation twice with 200 μl of washing solution, discard the filtrate; after centrifugation of the empty column, transfer the adsorption.

[0088] d. Transfer the column to a new tube, add 10 μl of DNA lysis buffer, and centrifuge to elute the DNA.

[0089] ②-3 Specific primer PCR amplification

[0090] ②-3.1 Preparation of the reaction system (operation on ice):

[0091] Prepare a reaction system with a total volume of 25 μl. The preparation method is as follows: Take 5 μl of sulfite-converted DNA as a template, add 4 μl of BCM primers and 12.5 μl of amplification enzyme, and finally add 3.5 μl of double-distilled water to the reaction tube to bring the total volume to 25 μl. After thorough mixing, briefly centrifuge, and place the reaction tube in a PCR instrument, ready to run the PCR reaction program.

[0092] ②-3.2 PCR program:

[0093] a. Initial high-temperature denaturation: Temperature set at 98℃, program runs for 2 minutes.

[0094] b. Gradient annealing loop (20 loops in total):

[0095] The first step of each cycle: set the temperature to 98℃ for denaturation, and run the program for 15 seconds.

[0096] The second step of each cycle: Set the temperature to annealing from 65℃, then decrease the temperature by 0.5℃ with each cycle until it reaches 55℃, running for a total of 20 cycles, with the duration matching the annealing requirements.

[0097] c. Constant Temperature Extension: Set the temperature to 65℃ and run continuously for 4 minutes.

[0098] d. Fixed annealing cycle (7 cycles in total):

[0099] The first step of each cycle: set the temperature to 98℃ for denaturation, and run the program for 15 seconds;

[0100] The second step of each cycle: set the temperature to 55℃ for annealing and extension, and run the program for 4 minutes.

[0101] e. Final extension: Set the temperature to 72℃ and run continuously for 5 minutes.

[0102] f. Low-temperature storage: Set the temperature to 4°C for continuous storage.

[0103] ②-3.3 Product purification:

[0104] a. Add 30 μl of magnetic beads to the product, mix well, incubate at room temperature for 5 minutes, and discard the supernatant after magnetic adsorption.

[0105] b. Wash the magnetic beads twice with 80% ethanol, air dry, add 13 μl of TE buffer, let stand for 5 minutes, centrifuge, and then transfer 10.5 μl of supernatant to a new tube.

[0106] ②-4 index primer PCR amplification

[0107] ①Addition to the reaction system:

[0108] Prepare a reaction system with a total volume of 25 μl. The preparation method is as follows: Take 10.5 μl of the product from the previous step as a template, add 2 μl of index primers, and finally add 12.5 μl of hot-start DNA polymerase to bring the total volume to 25 μl. After thorough mixing, briefly centrifuge, and place the reaction tube in a PCR instrument, ready to run the PCR reaction program.

[0109] ②PCR procedure:

[0110] a. Initial high-temperature denaturation: Set the temperature to 98℃ and run continuously for 1 minute.

[0111] b. Standard amplification cycle (12 cycles in total):

[0112] The first step of each cycle: set the temperature to 98℃ for denaturation, and run the program for 15 seconds.

[0113] The second step of each cycle: anneal at 60℃, run the program for 30 seconds.

[0114] The third step of each cycle: Set the temperature to 72℃ and extend the program for 30 seconds.

[0115] c. Final extension: Set the temperature to 72℃ and run continuously for 1 minute.

[0116] d. Low-temperature storage: Set the temperature to 4℃ and store continuously.

[0117] ③Final purification and preservation:

[0118] a. Add 25 μl of magnetic beads and mix well with the product. Incubate at room temperature for 5 minutes. After magnetic adsorption, discard the supernatant.

[0119] b. Wash the magnetic beads twice with 80% ethanol, air dry, add 30 μl ddH2O, let stand for 5 minutes, centrifuge, and then collect 30 μl of the supernatant and store at -40℃.

[0120] The above-mentioned method for constructing amplicon methylation libraries has the following innovations and advantages in detecting cfDNA methylation:

[0121] ① Innovative detection method: It innovatively combines multiplex PCR amplification and Bisulfite methylation sequencing, which can simultaneously detect several to hundreds of gene regions or sites.

[0122] ②Multiple detection sites: Enables rapid detection of methylation levels at numerous CpG sites in free DNA.

[0123] ③ Short experimental cycle: It can shorten the time required for detection.

[0124] ④ Low testing cost: Reduces testing expenses.

[0125] ③ High-throughput sequencing and data processing: After the library quality was deemed acceptable, high-throughput sequencing was performed using the Illumina platform. Sequencing data was filtered to remove adapters and low-quality sequences, and BSMAP software was used to align with the reference genome. The methylation status of the target gene region was accurately assessed by analyzing CpG site variation patterns.

[0126] In this embodiment of the invention, the concentration of TERT protein in serum is detected using a reagent kit.

[0127] The kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows: Human telomerase reverse transcriptase (TERT) calibrator and the sample to be tested are added to a microplate pre-coated with anti-human telomerase reverse transcriptase (TERT) antibody. Biotin-labeled anti-human telomerase reverse transcriptase antibody is added, mixed, and incubated. After incubation, the plate is washed, HRP-conjugated avidin is added, mixed, and incubated again. After incubation, the plate is washed to remove unbound components, forming a sandwich complex of solid-phase antibody-antigen-biotin-labeled antibody-avidinase on the solid phase surface. A chromogenic substrate (TMB chromogenic solution) is added; the reaction produces a blue product, which turns yellow after the reaction is stopped. The absorbance (OD value) is measured at 450 nm using a microplate reader; the absorbance is positively correlated with the TERT concentration in the sample. The concentration of TERT protein in the sample is calculated based on the calibration curve. The detection range of the kit is 0.312 ng / ml to 10 ng / ml.

[0128] The embodiments of the present invention have the following beneficial effects:

[0129] 1. In this embodiment of the invention, the two detection methods (TERT gene promoter methylation level detection and TERT protein concentration detection) are not the focus of this invention. The focus of this invention is to combine these two detection methods into a single scheme as a clinical prediction target, thereby achieving early warning of HBV-HCC. This invention uses serum for detection, which is readily available and does not require the use of patient liver tissue for detection as in other studies.

[0130] 2. In this embodiment of the invention, patients with chronic HBV infection were divided into four groups based on the disease progression trend: chronic hepatitis B (CHB), hepatitis B cirrhosis (LC), hepatitis B-related liver cancer (HBV-HCC), and non-HBV-HCC, plus a control group (i.e., healthy group). This resulted in a total of five groups. Theoretically, this verified the conclusion that "the higher the methylation level of the TERT gene promoter in serum, the higher the protein expression." Furthermore, considering the trends in each group, as the patient's condition worsened, the progression was from healthy individuals to chronic hepatitis B, then to hepatitis B cirrhosis, and finally to hepatitis B-related liver cancer. This progressively worsening trend validates the finding that "the higher the concentration of TERT protein in serum, the higher the methylation level of the TERT gene promoter."

[0131] Based on these two results, it can be concluded that in these two serum tests, as the patient's condition worsens—from chronic hepatitis B to hepatitis B cirrhosis, and then to hepatitis B-related liver cancer—the TERT gene promoter methylation level and TERT protein concentration gradually increase. Therefore, it can be assumed that if we test the serum TERT gene promoter methylation level and TERT protein concentration earlier, before the patient has progressed to hepatitis B cirrhosis or hepatitis B-related liver cancer, we can detect abnormalities in these indicators earlier. Elevated levels of these two indicators (serum TERT gene promoter methylation level and TERT protein concentration) allow for earlier intervention.

[0132] Clinical trial results showed that among 100 cases of chronic hepatitis B, all patients were taking medication and undergoing treatment as usual, and routine examinations were performed. The results showed that none of the 100 cases of chronic hepatitis B were showing a trend of worsening. However, using the combined detection method of this invention, the results showed that 20 cases had elevated serum TERT gene promoter methylation levels and TERT protein concentrations, indicating that the condition of these 20 cases was progressing towards worsening.

[0133] If patients with abnormalities in the above two indicators (serum TERT gene promoter methylation level and TERT protein concentration) receive normal treatment and follow-up, and undergo regular check-ups like other patients, but routine examinations cannot detect that these patients are progressing towards disease progression, then these two tests can screen for patients with this disease progression. For example, in patients with chronic hepatitis B, before they develop hepatitis B cirrhosis or hepatitis B-related liver cancer, early detection of these two abnormal indicators allows for early intervention or modification of their treatment plan. In this way, we can identify some patients with early-stage potential disease progression or malignant disease (which cannot be detected by routine examinations), and once these patients are identified, we can provide targeted treatment. For example, if a patient's medication is ineffective, we can adjust the medication in a timely manner, or if an early-stage tumor is detected, early treatment can be initiated.

[0134] Suppose we identify this type of patient, implement intervention and treatment, and then recheck these two indicators. If they show a decrease, it indicates that our intervention was effective, and the patient may have avoided developing malignant tumors. If, after intervention, these two indicators continue to rise, it suggests that the intervention method may not be the most effective for this patient, and we can try a different approach.

[0135] Therefore, through this method, we can screen out patients at risk of this condition. After screening, we can intervene in these types of patients and then determine whether the intervention is effective or not.

[0136] Even if all hepatitis B patients diligently take their medication and attend follow-up examinations as prescribed, current clinical testing indicators cannot achieve this predictive ability. The ultimate goal of this invention is to combine these two testing methods into a single clinical approach. This approach can serve as an assessment tool to evaluate the patient's disease progression and treatment effectiveness, thereby helping the patient control the disease's development.

[0137] In this embodiment of the invention, the reagent kit was purchased from Dongguan BioMedGene Technology Co., Ltd., product code: S10020, and its main components are as follows:

[0138] Note: Components in different batches of the kit are not interchangeable.

[0139] The reagent required but not provided in this embodiment of the invention is anhydrous ethanol (analytical grade).

[0140] Experimental verification scheme: In order to verify the effectiveness of the embodiments of the present invention in early warning of high-risk groups of HBV-HCC, we conducted relevant experimental verification.

[0141] The validation protocol was as follows: Individuals at different stages of HBV infection were selected and grouped for testing. Based on disease severity, they were divided into four groups: chronic hepatitis B (CHB), hepatitis B cirrhosis (LC), hepatitis B-related hepatocellular carcinoma (HBV-HCC), and non-HBV-HCC. A control group of healthy individuals was also selected. The methylation level of the TERT gene promoter in serum cf DNA and the concentration of serum TERT protein were measured in each group. The distribution trends of these indicators in each group were observed and analyzed.

[0142] Clinical trial verification:

[0143] The following groups were set up: Control group (blank group), CHB group (chronic hepatitis B group), LC group (hepatitis B cirrhosis group), HBV-HCC group (hepatitis B-related liver cancer group), and non-HBV-HCC group (non-hepatitis B-related liver cancer group).

[0144] Clinical Trial 1: 20 cases were included in the Control group, 20 cases in the CHB group, 20 cases in the LC group, 20 cases in the HBV-HCC group, and 20 cases in the non-HBV-HCC group. The methylation level of the TERT gene promoter in the serum cf DNA of each case was detected. The average value of the results of each group was calculated. See the attached table.

[0145] Clinical Trial 2: 60 cases were included in the Control group, 60 cases in the CHB group, 60 cases in the LC group, 60 cases in the HBV-HCC group, and 60 cases in the non-HBV-HCC group. The serum TERT protein concentration of each case was measured, and the average value of the results of each group was calculated. See Appendix Table 2.

[0146] Appendix 2: Mean values ​​of each group

[0147] The means of statistical values ​​for each experimental group were statistically analyzed using Prism 10.0 and SPSS 26.0 software. Quantitative data are expressed as mean ± standard deviation (x ± s); one-way ANOVA was used for pairwise comparisons between groups. The experimental results, after the above analysis, yielded... Figure 1 and Figure 2 .

[0148] Figure 1 and Figure 2 In the four groups (Control, CHB, LC, and HBV-HCC), the color of the bars changes from light to dark, representing the progression of the disease from mild to severe; the darker the color, the more severe the disease. The non-HBV-HCC group, serving as a control for the HBV-HCC group, is displayed separately as a patterned bar chart. P < 0.05 indicates a statistically significant difference. Figure 1 and Figure 2 In the diagram, * indicates P < 0.05. ** indicates P < 0.01. *** indicates P < 0.001.

[0149] Table 3 shows the results of one-way ANOVA for pairwise comparisons of TERT gene promoter methylation levels in serum cf DNA between groups:

[0150] Table 3

[0151]

[0152] Table 4 shows the results of one-way ANOVA for pairwise comparisons of serum TERT protein concentration among groups:

[0153] Table 4

[0154] In the above results, Figure 1 and Figure 2 The more asterisks (*) appear between groups, the smaller the p-value, indicating a more significant difference between the two groups. P < 0.05 indicates a statistically significant difference.

[0155] Results analysis:

[0156] The TERT gene promoter methylation level in serum cf DNA was lowest in healthy individuals. With disease progression, the methylation level increased sequentially in the CHB, LC, and HBV-HCC groups, indicating a close correlation between TERT gene promoter methylation level and the severity of chronic HBV infection. Although both are hepatocellular carcinoma, the TERT gene promoter methylation level was significantly higher in the HBV-HCC group than in the non-HBV-HCC group, further validating the specificity of TERT gene promoter methylation level in HBV-HCC. Similarly, serum TERT protein concentration was lowest in healthy individuals, gradually increasing in the CHB, LC, and HBV-HCC groups, suggesting that serum TERT protein concentration also increases with the progression of chronic HBV infection. Furthermore, as hepatocellular carcinoma, serum TERT protein concentration was significantly higher in the HBV-HCC group than in the non-HBV-HCC group, further demonstrating its specific elevation in HBV-HCC. These results indicate a close correlation between TERT gene promoter methylation level and serum TERT protein expression level. With increasing methylation levels of the TERT gene promoter, serum TERT protein expression also becomes more active, suggesting a potentially increased risk of hepatitis B patients progressing to HBV-HCC. Therefore, this experiment preliminarily validates the potential application value of the aforementioned combined detection protocol in early warning of HBV-HCC.

[0157] Patients with hepatitis B-related hepatocellular carcinoma (HBV-HCC) and patients with non-HBV-HCC also underwent these two tests (combined testing). The results showed a significant difference between HBV-HCC and non-HBV-HCC. This indicates that these two indicators (methylation level and protein concentration) are applicable for early warning of HBV-related hepatocellular carcinoma, but inaccurate and inapplicable for early warning of non-HBV-related hepatocellular carcinoma. Therefore, this invention is applied to the HBV-related population.

[0158] See Figure 3 ROC curve analysis of HBV-HCC patients, with combined detection including serum TERT gene promoter methylation level and TERT protein concentration as the core, showed that the combined detection can effectively predict and diagnose the progression of chronic HBV infection to hepatocellular carcinoma, with a sensitivity and specificity of 90.62% and 79.45%, respectively, while maintaining good diagnostic value (area under the ROC curve 0.927 (95% CI 0.859-0.969)). Its diagnostic value is significantly better than that of the common hepatocellular carcinoma marker AFP (area under the ROC curve 0.824 (95% CI 0.737-0.891)). Combined detection of serum TERT gene promoter methylation level and TERT protein concentration can effectively predict and diagnose the disease outcome in patients with chronic HBV infection.

[0159] according to Figure 3 The analysis yielded the following table:

[0160] Table 5

[0161] index sensitivity specificity AUC (95% CI) p value Joint testing 90.62 79.45 0.927（0.859-0.969） <0.0001 AFP 78.12 79.45 0.824（0.737-0.891） <0.0001

[0162] According to Table 5, a p-value < 0.0001 indicates a significant difference, which is statistically significant.

[0163] The innovation and applicability of this invention are as follows: This solution is designed based on the pathogenesis of hepatitis B-related liver cancer, and its core value lies in its unique detection specificity for this disease, which is a key feature distinguishing it from other detection solutions. Specifically, this solution is specifically designed for high-risk groups of hepatitis B-related liver cancer, providing targeted early warning and possessing special application value in disease prevention and intervention.

[0164] The following explains some of the terms / terms used in this invention:

[0165] Sensitivity: sensitivity;

[0166] Specificity: uniqueness;

[0167] AUC: Defined as the area under the ROC curve and the coordinate axis.

[0168] The ROC curve is essentially a curve plotted with FPR on the x-axis and TPR on the y-axis. The ROC curve reflects the relationship between sensitivity and specificity.

[0169] Explanation of CpG sites:

[0170] CpG sites refer to dinucleotide sequences (i.e., "CpG") formed by the linkage of cytosine (C) and guanine (G) in genomic DNA via phosphodiester bonds (p). In the human genome, these sequences often cluster together to form CpG islands, typically located in gene promoter regions or near the first exon. CpG sites are core units of epigenetic regulation, and their methylation status is closely related to gene expression, cellular function, and disease development.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of combined detection of methylation level and protein concentration in early warning of HBV-HCC, characterized in that, The combined detection of serum TERT gene promoter methylation level and TERT protein concentration was applied to the early warning of hepatitis B virus-related hepatocellular carcinoma.

2. The application of the combined detection of methylation level and protein concentration in early warning of HBV-HCC according to claim 1, characterized in that, The combined detection of TERT gene promoter methylation level and TERT protein concentration specifically involves combining two detection methods—detection of TERT gene promoter methylation level in serum cell-free DNA and detection of serum TERT protein concentration—into a single scheme as a clinical prediction target.

3. The application of the combined detection of methylation level and protein concentration in early warning of HBV-HCC according to claim 2, characterized in that, The detection of TERT gene promoter methylation level in serum free DNA includes the following steps: (1) Collect serum and perform plasma separation; (2) Extraction of cell-free DNA; (3) Detection of methylation level.

4. The application of the combined detection of methylation level and protein concentration according to claim 3 in early warning of HBV-HCC, characterized in that, The plasma separation was performed using a two-step centrifugation method: The serum was centrifuged at a low speed, and the first supernatant was collected into a low-adsorption tube. Then, the first supernatant is centrifuged at a high speed, and the second supernatant is collected. The second supernatant is then transferred, aliquoted, and stored to avoid aspirating white blood cells or red blood cells.

5. The application of the combined detection of methylation level and protein concentration according to claim 3 in early warning of HBV-HCC, characterized in that, The extraction of free DNA includes the following steps: a. Add protease lysis buffer to dissolve the protease. Add the dissolved protease, magnetic beads and plasma sample to a centrifuge tube, mix well, add lysis buffer, mix well and vortex. b. After centrifugation and sedimentation, place the centrifuge tube on a magnetic rack for adsorption and discard the third supernatant; c. Add the first and second washing solutions sequentially, shaking to mix well after each addition, then adsorb and discard the fourth supernatant. d. Repeat the second washing solution, shake to mix, adsorb, discard the residual liquid, and air dry at room temperature; e. Add the elution buffer, vortex to mix, and then vortex again. f. After placing the centrifuge tube on a magnetic rack for adsorption, collect the supernatant, i.e., the free DNA solution, and store it below -10°C.

6. The application of the combined detection of methylation level and protein concentration according to claim 3 in early warning of HBV-HCC, characterized in that, The methylation level detection employs amplicon methylation sequencing (Multiplex PCR Bisulfite Sequencing, MultiplexBS) combined with bisulfite treatment and multiplex PCR amplification. The amplicon methylation sequencing technology innovatively combines multiplex PCR amplification and bisulfite methylation sequencing, enabling the simultaneous detection of several to hundreds of gene regions or sites. Specifically, it includes the following steps: DNA sample testing; Construction of amplicon methylation libraries; High-throughput sequencing and data processing.

7. The application of the combined detection of methylation level and protein concentration according to claim 6 in early warning of HBV-HCC, characterized in that, The steps DNA sample testing includes: a. Use agarose gel electrophoresis to detect the degree of DNA degradation and contaminant bands; b. Use Qubit 3.0 to quantify DNA concentration, ensuring that the DNA concentration is ≥5ng / μL and the total amount is ≥50ng for library construction.

8. The application of the combined detection of methylation level and protein concentration according to claim 6 in early warning of HBV-HCC, characterized in that, The steps The construction of amplicon methylation libraries includes: Design of TERT gene promoter region-specific primer sequences; Sulfite treatment of free DNA; PCR amplification using specific primers; PCR amplification using index primers.

9. The application of the combined detection of methylation level and protein concentration according to claim 6 in early warning of HBV-HCC, characterized in that, The steps High-throughput sequencing and data processing include: a. After the library quality is qualified, high-throughput sequencing is performed using the Illumina platform; b. Filter the sequencing data to remove adapters and low-quality sequences, and use BSMAP software to align with the reference genome; c. By analyzing the variation patterns of CpG sites, the methylation status of the target gene region can be accurately assessed.

10. The application of the combined detection of methylation level and protein concentration according to claim 2 in early warning of HBV-HCC, characterized in that, The serum TERT protein concentration was determined using a kit that employed a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows: a. Add human telomerase reverse transcriptase (TERT) calibrator and the sample to be tested to a microplate pre-coated with anti-human telomerase reverse transcriptase (TERT) antibody; b. Add biotin-labeled anti-human telomerase reverse transcriptase antibody, mix well and incubate; c. After incubation, wash, add HRP-conjugated avidin, mix well, and incubate again; d. After incubation, wash to remove unbound components and form a sandwich complex of solid-phase antibody-antigen-biotin-labeled antibody-avidinase on the solid-phase surface; e. Add the chromogenic substrate (TMB chromogenic solution), the reaction produces a blue product, which turns yellow after the reaction is terminated; f. Measure the absorbance (OD value) at a wavelength of 450 nm using an ELISA reader. The absorbance is positively correlated with the TERT concentration in the sample. g. Calculate the concentration of TERT protein in the sample based on the calibration curve. The kit's detection range is 0.312 ng / ml - 10 ng / ml.

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