A nasopharyngeal carcinoma biomarker and application thereof

Abstract

The present application relates to the technical field of medical examination, in particular to a nasopharyngeal carcinoma biomarker and application thereof. The present application finds that the overall antibody levels of BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG and BLLF3-IgA in serum of nasopharyngeal carcinoma patients are significantly higher than those of nasopharyngeal benign disease controls and healthy controls. Further analysis finds that the combination of the five antibodies can replace the combination of VCA-IgA antibody and EBNA1-IgA, and can be effectively used for the diagnosis of nasopharyngeal carcinoma, and can overcome the problems of strict biological safety conditions required in the production process of EBV-VCA natural protein and poor reagent stability between batches. In addition, the present application also finds that the greater the disease-free survival probability of nasopharyngeal carcinoma patients with low BLLF3-IgA and BDLF1-IgA antibody levels, which indicates the potential value of the two markers in the prognosis evaluation of nasopharyngeal carcinoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical technology, in particular to a nasopharyngeal carcinoma biomarker, in particular BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, BLLF3-IgA, and application thereof. BACKGROUND

[0002] Nasopharyngeal carcinoma (NPC) is a malignant tumor occurring in the epithelium and crypt columnar epithelium of the nasopharyngeal mucosa. In high-incidence areas, nasopharyngeal carcinoma is mostly low-differentiated squamous carcinoma. The disease has a high sensitivity to radiotherapy, and thus radiotherapy is often used as the main clinical treatment method. Studies have shown that the five-year survival rate of nasopharyngeal carcinoma patients in the early stage is significantly higher than that of nasopharyngeal carcinoma patients in the advanced stage. However, due to the relatively concealed location of nasopharyngeal carcinoma, the early symptoms are not obvious, and most patients are diagnosed in the middle and advanced stages.

[0003] Epstein-Barr virus (EBV), also known as human herpesvirus 4 (HHV4), is the first virus confirmed to be associated with human tumorigenesis. Studies have shown that EBV infection is associated with Burkitt's lymphoma, NK cell lymphoma Hodgkin's lymphoma, T cell lymphoma and nasopharyngeal carcinoma and other malignant tumors. Among them, the occurrence of nasopharyngeal carcinoma is most closely related to EBV infection. Studies have found that EBV can be detected in almost all undifferentiated NPC tumor cells. Given the close relationship between EBV infection and the formation and development of nasopharyngeal carcinoma, EBV-related antibody markers such as EBV capsid antigen (VCA)-IgA, Rta-IgA, early antigen (EA)-IgA, and EBV nuclear antigen-1 (EBNA1)-IgA are often used to diagnose and screen nasopharyngeal carcinoma. These means have the advantages of non-invasiveness, patient compliance, etc., and are conducive to the early diagnosis, prognosis and efficacy evaluation of nasopharyngeal carcinoma in high-incidence areas. However, these markers still cannot fully meet the current clinical detection needs. For example, EBV capsid antigen VCA is a multimeric protein composed of BFRF3 (p18), BLRF2 (p23), BDRF1 (p40), BNRF1 (p143), BCLF1 (p150), BALF4 (gp125) and other proteins, which is not easy to express completely in vitro. Currently, there are two forms of VCA coating antigens used in EBV VCA-IgA antibody detection kits: (1) VCA natural protein purified from EB virus-infected cell lysate, and (2) combined use of multiple VAC component recombinant proteins. Studies have found that the sensitivity of the VCA-IgA antibody detection kit using VCA natural protein as the coating antigen is significantly higher than that of the VCA-IgA antibody detection kit using recombinant protein as the coating antigen. However, strict biosafety conditions are required during the production of VCA natural protein, and the consistency of reagent detection results between different manufacturers and the stability of reagents between batches still need to be solved. Therefore, screening and identifying virus-related serum antibody markers with better specificity and sensitivity, and easy to prepare and standardize, is still a problem to be solved. SUMMARY

[0004] To overcome the deficiencies of the prior art, the present application provides a nasopharyngeal carcinoma biomarker, in particular BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, BLLF3-IgA, and applications thereof.

[0005] In the first aspect of the present application, the use of a combination of one or more of BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, and BLLF3-IgA as markers in the preparation of products for the diagnosis, prevention, treatment, severity assessment, efficacy evaluation, and drug screening of nasopharyngeal carcinoma is provided.

[0006] Specifically, the above-mentioned immunoglobulin is used as a sole marker or in combination with other markers as a companion diagnostic marker; in some embodiments of the present application, the other marker is EBNA1-IgA.

[0007] In some embodiments of the present application, the marker is a combination of BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG and BLLF3-IgA.

[0008] In some embodiments of the present application, the marker is a combination of BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, BLLF3-IgA and EBNA1-IgA.

[0009] In some embodiments of the present application, the application is the use of the above-mentioned immunoglobulin as a marker in the preparation of a diagnostic product for nasopharyngeal carcinoma.

[0010] In some embodiments of the present application, the application is the use of the above-mentioned immunoglobulin as a marker in the preparation of a diagnostic product for nasopharyngeal carcinoma.

[0011] In some embodiments of the present application, the application is the use of the above-mentioned immunoglobulin as a marker in the preparation of a diagnostic product for nasopharyngeal carcinoma.

[0012] Specifically, in the above-mentioned application, the severity assessment of nasopharyngeal carcinoma is to determine whether it is in early stage (stage I and stage II) or in advanced stage (stage III and stage IV).

[0013] In some embodiments of the present application, the application is the use of BLRF2-IgA and / or BDLF1-IgG (particularly BLRF2-IgA) as a marker in the preparation of a diagnostic product for the severity assessment of nasopharyngeal carcinoma. When the level of BLRF2-IgA and / or BDLF1-IgG (particularly BLRF2-IgA) in the sample is high, the probability of the nasopharyngeal carcinoma of the subject being in advanced stage is high.

[0014] Specifically, the above-mentioned product can be a reagent, a kit, a test paper, a chip, etc., which can contain a substance capable of binding to the antibody protein (such as an antibody or a fragment thereof); or an instrument platform, such as a proteomics analysis product; the proteomics analysis product can contain a measurement module (for measuring the content of the antibody protein in the sample to be tested) and an analysis module (for analyzing the difference in the content of the antibody protein between the sample to be tested and a reference sample).

[0015] Specifically, the test samples for the above-mentioned products can be, for example, blood (especially peripheral blood) or fractions thereof (e.g., serum, plasma) obtained from biopsy subjects, especially serum.

[0016] In a second aspect of the invention, a reagent for detecting one or more combinations of BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, and BLLF3-IgA is provided for use in the preparation of products for the diagnosis, prevention, treatment, severity assessment, efficacy evaluation, and drug screening of nasopharyngeal carcinoma.

[0017] In some embodiments of the present invention, the application is the use of reagents for detecting the above-mentioned immunoglobulins in the preparation of diagnostic products for nasopharyngeal carcinoma.

[0018] In one embodiment of the present invention, the application is the use of a reagent for detecting a combination of BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, BLLF3-IgA and EBNA1-IgA in the preparation of a diagnostic product for nasopharyngeal carcinoma.

[0019] In some embodiments of the present invention, the application is the use of reagents for detecting the above-mentioned immunoglobulins in the preparation of products for assessing the severity of nasopharyngeal carcinoma.

[0020] Specifically, in the above applications, the severity of the nasopharyngeal carcinoma is assessed by determining whether it is early (stage I and II) or late (stage III and IV).

[0021] In some embodiments of the invention, the application is the use of reagents for detecting BLRF2-IgA and / or BDLF1-IgG (especially BLRF2-IgA) in the preparation of products for assessing the severity of nasopharyngeal carcinoma. Higher levels of BLRF2-IgA and / or BDLF1-IgG (especially BLRF2-IgA) in the test sample indicate a higher probability that the subject has advanced-stage nasopharyngeal carcinoma.

[0022] Specifically, the detection can be qualitative or quantitative, especially quantitative.

[0023] Specifically, the detection reagent may include substances capable of binding the aforementioned immunoglobulins (e.g., antibodies or fragments thereof).

[0024] Specifically, reagents for detecting the aforementioned immunoglobulins can be based on known methods for using proteins to perform their functions: for example, ELISA, Western blotting, indirect immunofluorescence, enzyme immunospot assay, immunoluminescence assay, radioimmunoassay, immunohistochemistry, Western blotting, proteomics (e.g., antibody microarrays, mass spectrometry (e.g., data independent acquisition (DIA) mass spectrometry)) can be used.

[0025] Specifically, samples used for immunoglobulin detection can be, for example, blood (especially peripheral blood) or fractions thereof (e.g., serum, plasma) obtained from a biopsy subject, especially serum.

[0026] Specifically, the aforementioned products can be reagents, kits, test strips, chips, proteomics analysis products (such as antibody chips, DIA-MS), etc.

[0027] In a third aspect of the invention, BLLF3-IgA and / or BDLF1-IgA (especially BDLF1-IgA) are provided as biomarkers for use in the preparation of nasopharyngeal carcinoma prognostic products.

[0028] Specifically, the prognosis includes the prediction of the probability of disease-free survival (DFS) for patients with nasopharyngeal carcinoma, such as the prediction of a DFS probability exceeding 0.75, 0.8, 0.85, 0.9, 0.95, or 0.99.

[0029] Specifically, when BLLF3-IgA and / or BDLF1-IgA levels are low, the probability of DFS in nasopharyngeal carcinoma patients is higher than 0.9, 0.95, or even 0.99.

[0030] In some embodiments of the present invention, when BLLF3-IgA levels are low, the probability of DFS in nasopharyngeal carcinoma patients is higher than 0.9, 0.95, or even 0.99.

[0031] In some embodiments of the present invention, when the BLLF3-IgA level is high, the probability of DFS in nasopharyngeal carcinoma patients is lower than 0.85, and the probability of 0.8 is relatively high.

[0032] In some embodiments of the present invention, when BDLF1-IgA levels are low, the probability of a disease-free survival (DFS) rate exceeding 0.9 is relatively high in nasopharyngeal carcinoma patients.

[0033] In some embodiments of the present invention, when BDLF1-IgA levels are high, the probability of a disease-free survival (DFS) probability of nasopharyngeal carcinoma patients being less than 0.8 is relatively high.

[0034] Specifically, the aforementioned products can be reagents, kits, test strips, chips, etc., which may contain substances capable of binding antibody proteins (such as antibodies or fragments thereof); they can also be instrument platforms, such as proteomics analysis products; proteomics analysis products may include a measurement module (for measuring the content of antibody proteins in the test sample) and an analysis module (for analyzing the difference in the content of antibody proteins between the test sample and the reference sample).

[0035] Specifically, the test samples for the above-mentioned products can be, for example, blood (especially peripheral blood) or fractions thereof (e.g., serum, plasma) obtained from biopsy subjects, especially serum.

[0036] In a fourth aspect of the invention, the use of reagents for detecting BLLF3-IgA and / or BDLF1-IgA (especially BDLF1-IgA) in the preparation of nasopharyngeal carcinoma prognostic products is provided.

[0037] Specifically, the prognosis includes the prediction of the probability of disease-free survival (DFS) for patients with nasopharyngeal carcinoma, such as the prediction of a DFS probability exceeding 0.8, 0.85, 0.9, 0.95, or 0.99.

[0038] Specifically, when BLLF3-IgA and / or BDLF1-IgA levels are low, the probability of DFS in nasopharyngeal carcinoma patients is higher than 0.9, 0.95, or even 0.99.

[0039] In some embodiments of the present invention, when BLLF3-IgA levels are low, the probability of DFS in nasopharyngeal carcinoma patients is higher than 0.9, 0.95, or even 0.99.

[0040] In some embodiments of the present invention, when the BLLF3-IgA level is high, the probability of DFS in nasopharyngeal carcinoma patients is lower than 0.85, and the probability of 0.8 is relatively high.

[0041] In some embodiments of the present invention, when BDLF1-IgA levels are low, the probability of a disease-free survival (DFS) rate exceeding 0.9 is relatively high in nasopharyngeal carcinoma patients.

[0042] In some embodiments of the present invention, when BDLF1-IgA levels are high, the probability of a disease-free survival (DFS) probability of nasopharyngeal carcinoma patients being less than 0.8 is relatively high.

[0043] Specifically, the detection can be qualitative or quantitative, especially quantitative.

[0044] Specifically, the detection reagent may include substances (e.g., antibodies or fragments thereof) capable of binding BLLF3-IgA and / or BDLF1-IgA.

[0045] Specifically, reagents for detecting BLLF3-IgA and / or BDLF1-IgA can function based on known methods using the proteins: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, proteomics (e.g., antibody microarrays), mass spectrometry (e.g., data independent acquisition (DIA) mass spectrometry), etc.

[0046] Specifically, samples used for BLLF3-IgA and / or BDLF1-IgA detection can be, for example, blood (especially peripheral blood) or fractions thereof (e.g., serum, plasma) obtained from a biopsy subject, especially serum.

[0047] Specifically, the aforementioned products can be reagents, kits, test strips, chips, proteomics analysis products (such as antibody chips, DIA-MS), etc.

[0048] In a fifth aspect of the invention, the use of reagents that affect the level or activity of BLLF3-IgA and / or BDLF1-IgA (especially BDLF1-IgA) in the preparation of products for the prevention, treatment, and efficacy evaluation of nasopharyngeal carcinoma is provided.

[0049] Specifically, the effect is to reduce the levels or protein activity of BLLF3-IgA and / or BDLF1-IgA in the subjects, particularly in serum, which is beneficial to improving the probability of disease-free survival (DFS) in patients with nasopharyngeal carcinoma.

[0050] Specifically, the reagents affecting BLLF3-IgA and BDLF1-IgA are BLLF3-IgA and BDLF1-IgA inhibitors, which can reduce the level or protein activity of BLLF3-IgA and / or BDLF1-IgA in the subject's body (especially in serum).

[0051] In some embodiments of the present invention, the above-mentioned product is a pharmaceutical preparation used for the prevention or treatment of nasopharyngeal carcinoma.

[0052] Specifically, the aforementioned pharmaceutical preparations may also include one or more pharmaceutically acceptable excipients, such as fillers, binders, humectants, disintegrants, lubricants, flavorings, sweeteners, antioxidants, preservatives, etc.

[0053] Specifically, the aforementioned pharmaceutical preparations can be any suitable dosage form, such as oral dosage forms (e.g., tablets, pills, powders, granules, capsules, etc.), injections (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), and topical dosage forms (e.g., ointments, creams, lotions, film-forming preparations, etc.), especially oral dosage forms and injections.

[0054] Specifically, the various dosage forms mentioned above can be prepared according to conventional pharmaceutical manufacturing methods. For example, the active ingredient can be mixed with one or more pharmaceutically acceptable excipients and then formulated into the desired dosage form.

[0055] In a sixth aspect of the invention, the use of BLLF3-IgA and / or BDLF1-IgA (especially BDLF1-IgA) inhibitors in the preparation of medicaments for the prevention and / or treatment of nasopharyngeal carcinoma is provided.

[0056] Specifically, the inhibitor can be a small molecule compound or a pharmaceutically acceptable salt thereof, or a macromolecule such as an antibody.

[0057] Specifically, the drug may be in any suitable dosage form, such as oral dosage forms (e.g., tablets, pills, powders, granules, capsules, etc.), injectable dosage forms (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), and topical dosage forms (e.g., ointments, creams, lotions, film-forming preparations, etc.), especially oral dosage forms and injectable dosage forms.

[0058] In a seventh aspect of the present invention, a product for the diagnosis, severity assessment, efficacy evaluation, and drug screening of nasopharyngeal carcinoma is provided, comprising a reagent for detecting one or more of BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, and BLLF3-IgA.

[0059] Specifically, the product also includes reagents for detecting other markers such as EBNA1-IgA.

[0060] Specifically, the products may be reagents, kits, test strips, gene chips, proteomics analysis products (such as antibody chips, DIA-MS), etc.

[0061] In an eighth aspect of the invention, a prognostic product for nasopharyngeal carcinoma is provided, comprising reagents for detecting BLLF3-IgA and / or BDLF1-IgA (particularly BDLF1-IgA).

[0062] Specifically, the products may be reagents, kits, test strips, gene chips, proteomics analysis products (such as antibody chips, DIA-MS), etc.

[0063] In a ninth aspect of the invention, a medicament for the prevention and / or treatment of nasopharyngeal carcinoma is provided, comprising agents that affect the levels or activity of BLLF3-IgA and / or BDLF1-IgA (particularly BDLF1-IgA).

[0064] Specifically, the effect is to reduce the levels or protein activity of BLLF3-IgA and / or BDLF1-IgA in the subjects, particularly in serum, which is beneficial to improving the disease-free survival (DFS) rate of nasopharyngeal carcinoma patients.

[0065] Specifically, the reagents affecting BLLF3-IgA and BDLF1-IgA are BLLF3-IgA and BDLF1-IgA inhibitors, which can reduce the level or protein activity of BLLF3-IgA and / or BDLF1-IgA in the subject's body (especially in serum).

[0066] In a tenth aspect of the present invention, a method for diagnosing nasopharyngeal carcinoma is provided, comprising the step of detecting one or more of BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, and BLLF3-IgA.

[0067] Specifically, the method may include the following steps:

[0068] (1) Obtaining subject samples;

[0069] (2) Detect the levels of the above-mentioned immunoglobulins in the subject samples;

[0070] (3) Link the test results to whether the subject has the disease or the risk of disease.

[0071] In particular, elevated levels of the immunoglobulins compared to normal controls indicate a high risk of being diagnosed with nasopharyngeal carcinoma or having nasopharyngeal carcinoma in the subject. It should be noted that the specific risk and severity of the disease require a comprehensive assessment by a clinician, considering other relevant test results.

[0072] Specifically, the subjects were humans.

[0073] Specifically, the sample may be, for example, blood (particularly peripheral blood) or fractions thereof (e.g., serum, plasma) obtained from a biopsy subject, particularly serum.

[0074] In an eleventh aspect of the present invention, a method for predicting the prognosis of nasopharyngeal carcinoma is provided, comprising the step of detecting BLLF3-IgA and / or BDLF1-IgA (especially BDLF1-IgA).

[0075] Specifically, the method may include the following steps:

[0076] (1) Obtaining subject samples;

[0077] (2) Detect the levels of the above-mentioned immunoglobulins in the subject samples;

[0078] (3) Link the test results with the prognosis of the subjects.

[0079] In particular, if the level of the immunoglobulin is elevated compared to normal controls, the subject is identified as having a poor prognosis (low probability of disease-free survival). It should be noted that the specific prognosis of the disease requires a comprehensive assessment by clinicians, taking into account other test results of the subject.

[0080] Specifically, the subjects were humans.

[0081] Specifically, the sample may be, for example, blood (particularly peripheral blood) or fractions thereof (e.g., serum, plasma) obtained from a biopsy subject, particularly serum.

[0082] The inventors of this invention discovered through ELISA detection that the overall antibody levels of five biomarker antibodies (BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, and BLLF3-IgA) and EBNA1-IgA in the serum of nasopharyngeal carcinoma patients were significantly higher than those in controls with benign nasopharyngeal diseases and healthy controls (P<0.05). Furthermore, most of these antibodies were also present at high levels in the serum of early-stage nasopharyngeal carcinoma patients. Further logistic regression analysis revealed that the five-antibody combination can replace the VCA-IgA antibody, and its combined use with the EBNA1-IgA antibody is effective for the diagnosis of nasopharyngeal carcinoma, overcoming the problems of strict biosafety conditions and poor batch-to-batch reagent stability in the production of EBV-VCA natural protein. In addition, the inventors found that nasopharyngeal carcinoma patients with high levels of BLLF3-IgA and BDLF1-IgA antibodies had longer disease-free survival, indicating the potential value of these two biomarker antibodies in the prognostic assessment of nasopharyngeal carcinoma. Attached Figure Description

[0083] Figure 1 The figure shows a comparison of the levels of antibodies BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, BLLF3-IgA, and EBNA1-IgA in cohort 1 detected by ELISA.

[0084] Figure 2 The results show the sensitivity and specificity of the marker antibody in distinguishing between nasopharyngeal carcinoma patients, benign nasopharyngeal disease control groups, and healthy controls.

[0085] Figure 3 The figure shows the survival analysis of biomarker antibodies and disease-free survival. Detailed Implementation

[0086] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0087] According to the TNM staging system, nasopharyngeal carcinoma is typically divided into stages I, II, III, and IV, representing the stages from early to late stages. Stage I: The tumor is confined to the nasopharynx or invades the oropharynx or nasal cavity, without involvement of the parapharyngeal space, regional lymph node metastasis, or distant metastasis. Stage II: The tumor invades the parapharyngeal space and adjacent soft tissues, with unilateral cervical lymph node metastasis, but no distant metastasis. Stage III: The tumor invades the skull base, cervical pterygoid structures, and parasinusoids, with bilateral lymph node metastasis, the largest diameter ≤6cm, above the level of the cricoid cartilage, and no distant metastasis. Tumor invasion of intracranial cranial nerves, hypopharynx, infratemporal fossa, and orbit is classified as stage IV. Stage IVa: The tumor invades the intracranial space and involves cranial nerves, with extensive soft tissue infiltration of the hypopharynx, orbit, and parotid gland extending beyond the lateral pterygoid muscle, unilateral and bilateral lymph node metastasis with a largest diameter >6cm, but no distant metastasis. Stage IVb: The tumor has metastasized to distant sites. Staging is usually determined by enhanced MRI of the nasopharynx and neck, enhanced CT of the chest, CT of the upper abdomen, whole-body bone scan, or PET-CT. In this invention, stages I and II are considered early stages, and stages III and IV are considered late stages.

[0088] Immunoglobulins are animal proteins with antibody activity. They are mainly found in blood plasma, but also in other body fluids, tissues, and some secretions. Immunoglobulins can be classified into five classes: IgG, IgA, IgM, IgD, and IgE.

[0089] In this invention, "diagnosing nasopharyngeal carcinoma" includes both determining whether the subject already has nasopharyngeal carcinoma and determining whether the subject is at risk of developing nasopharyngeal carcinoma.

[0090] In this invention, "prognosis" refers to the prediction of the disease's progression and consequences. Based on whether treatment is received during the disease's occurrence or development, prognosis can be divided into natural prognosis and therapeutic prognosis. Natural prognosis is the prediction of the disease's progression and consequences without treatment; therapeutic prognosis is the prediction of the disease's progression and final consequences through medical intervention. The prognosis described in this invention generally refers to therapeutic prognosis. For example, the prognosis described in this invention includes the prediction of the disease-free survival (DFS) probability for nasopharyngeal carcinoma patients, such as a prediction that the DFS probability (e.g., within 10, 8, 7, 6, 5, 4, 3, 2, or 1 year after treatment) exceeds 0.75, 0.8, 0.85, 0.9, 0.95, or 0.99.

[0091] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.

[0092] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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.

[0093] Example 1

[0094] 1. Sample collection

[0095] Samples were collected from the Second Affiliated Hospital of Guangzhou Medical University, the Cancer Center of Guangzhou Medical University, and the Cancer Center of Sun Yat-sen University. Informed consent was obtained from all patients. A total of 1,665 serum samples were collected, including: serum from patients with nasopharyngeal carcinoma (early stage (I and II) and late stage (III and IV)), serum from patients with benign nasopharyngeal diseases, and serum from healthy controls.

[0096] Based on the different sources of nasopharyngeal carcinoma samples, they were divided into three clinical cohorts.

[0097] The Cohort 1 sample consisted of 594 cases, all from the Second Affiliated Hospital of Guangzhou Medical University. It included serum samples from 240 patients with nasopharyngeal carcinoma (54 early-stage samples and 156 late-stage samples), serum samples from 254 patients with benign nasopharyngeal diseases, and serum samples from 100 healthy individuals.

[0098] The Cohort 2 sample consisted of 774 cases, including 501 serum samples from nasopharyngeal carcinoma patients (from Sun Yat-sen University Cancer Center, including 61 early-stage samples and 433 late-stage samples), 111 serum samples from patients with benign nasopharyngeal diseases (from Sun Yat-sen University Cancer Center and the Second Affiliated Hospital of Guangzhou Medical University), and 162 serum samples from healthy individuals (from Sun Yat-sen University Cancer Center).

[0099] The Cohort 3 sample consisted of 297 cases, including 108 serum samples from nasopharyngeal carcinoma patients (from the Cancer Hospital of Guangzhou Medical University, including 16 early-stage samples and 88 late-stage samples), 100 serum samples from patients with benign nasopharyngeal diseases (from the Second Affiliated Hospital of Guangzhou Medical University), and 89 serum samples from healthy individuals (from the Second Affiliated Hospital of Guangzhou Medical University).

[0100] See the table below for details:

[0101] Table 1. Sample Source Information for Cohort 1

[0102]

[0103] Table 2. Sample Source Information for Cohort 2

[0104]

[0105] Table 3. Sample Source Information for Cohort 3

[0106]

[0107] 2. Detection Method

[0108] To assess the concentration of serum marker antibodies, an enzyme-linked immunosorbent assay (ELISA) is performed. The specific steps are as follows:

[0109] (1) Viral protein coating: The candidate marker protein after expression and purification was diluted with ELISA coating solution (prepared by our laboratory, the main component of which is 0.05M carbonate buffer with pH 9.6), added to the microwell of the ELISA plate, sealed with the sealing film, and incubated overnight at 4°C.

[0110] (2) Discard the coating solution, wash the plate with PBST, pat dry, add 5% milk, seal the plate with the sealing film, and incubate at 37°C for 1 hour.

[0111] (3) Discard the liquid in the ELISA plate, wash the plate with PBST, and pat dry. Add the serum sample diluted with 5% milk and incubate at 37°C for 1 hour.

[0112] (4) Discard the liquid in the ELISA plate, wash the plate with PBST, and pat dry. Add HRP-labeled anti-human IgG and IgA antibodies diluted with 5% milk (Jackson Immuno Research), and incubate at 37°C for 1 hour.

[0113] (5) Wash the plate with PBST and pat dry. Mix TMB-A and 1×TMB from the TMB substrate chromogenic kit (Kangwei Century CW0050S) at a ratio of 1:19, add to the ELISA plate, incubate at room temperature in the dark, then add ELISA stop solution (Beijing Solarbio C1058) to terminate the reaction. Place the ELISA plate in a microplate reader and detect at a wavelength of 450 nm.

[0114] The VCA-IgA antibody in serum samples was detected according to the instructions of the anti-EB virus capsid antigen antibody IgA detection kit (enzyme-linked immunosorbent assay, purchased from Euromon Medical Diagnostics (China) Co., Ltd.).

[0115] 3. Statistical Analysis

[0116] Differences in ELISA results were compared using the t-test in GraphPad Prism software, with p < 0.05 indicating statistical significance. Using the ELISA results of Cohort 2 as the training set and the ELISA results of Cohort 1 and Cohort 3 as the test set, logistic regression analysis was performed to model the biomarker antibodies. Based on disease-free survival (DFS) information of nasopharyngeal carcinoma patients, the Kaplan-Meier method was used to analyze the prognostic value of biomarker antibodies for nasopharyngeal carcinoma, and survival differences were analyzed using the Log-rank test.

[0117] 4. Experimental Results

[0118] The expression levels of various biomarker antibodies in serum samples from patients with nasopharyngeal carcinoma, patients with benign nasopharyngeal diseases, and healthy individuals in Cohort 1 were analyzed using ELISA, and the results are expressed as absorbance at 450 nm. See attached figures. Figure 1 As shown, the levels of marker antibodies such as BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, BLLF3-IgA, and EBNA1-IgA were significantly higher in the serum of both early-stage nasopharyngeal carcinoma (NPC) patients and advanced-stage NPC patients than in the control group (P<0.05).

[0119] Based on the ELISA results of each biomarker antibody in three validation cohorts, we plotted receiver operating characteristic (ROC) curves to further analyze the ability of each biomarker antibody to distinguish between nasopharyngeal carcinoma (NPC) patients and healthy controls, as well as between NPC patients and benign nasopharyngeal disease controls. See results below. Figure 2 Under 95% specificity conditions, the antibody combination of 5 antibodies (BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, and BLLF3-IgA) + EBNA1-IgA showed similar sensitivity in distinguishing nasopharyngeal carcinoma patients from healthy controls and benign disease controls to the known biomarker combination VCA-IgA + EBNA1-IgA. This indicates that 5 antibodies can replace VCA-IgA antibodies, and their combination with EBNA1-IgA antibodies can be effectively used for the diagnosis of nasopharyngeal carcinoma. Furthermore, it can overcome the problems of requiring strict biosafety conditions and poor batch-to-batch reagent stability in the production of EBV-VCA natural protein.

[0120] To evaluate the prognostic value of the biomarker antibodies screened in this study for nasopharyngeal carcinoma, we followed up on the disease progression and prognostic information of 136 nasopharyngeal carcinoma patients in Cohort 1. We divided the ELISA results of each biomarker antibody into two groups: high (top 25%) and low (bottom 25%). Survival analysis was performed on these two groups to compare disease-free survival (DFS). Results are as follows: Figure 3 As shown, nasopharyngeal carcinoma patients with low levels of BLLF3-IgA and BDLF1-IgA antibodies had a higher probability of DFS (P values ​​were 0.035 and 0.0056, respectively), demonstrating the potential value of these two biomarker antibodies in prognostic assessment of nasopharyngeal carcinoma.

[0121] 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, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0122] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0123] Listing the steps of the method in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. The application of reagents for detecting biomarkers in the preparation of diagnostic products for nasopharyngeal carcinoma, characterized in that, The biomarkers are a combination of BLRF2-IgA, BLRF2-IgG, BDLF1-IgA, BDLF1-IgG, BLLF3-IgA, and EBNA1-IgA.

2. The application as described in claim 1, characterized in that, The products mentioned are reagents, kits, test strips, chips, or proteomics analysis products.

3. The application as described in claim 1 or 2, characterized in that, The test sample for the product is blood or its fraction obtained from a self-biopsy subject.

4. The application as described in claim 1 or 2, characterized in that, The test sample for the product was serum.

5. The application of reagents for detecting biomarkers in the preparation of prognostic products for nasopharyngeal carcinoma, characterized in that, The biomarker is BDLF1-IgA, or a combination of BDLF1-IgA and BLLF3-IgA, and the prognosis is a prediction of the probability of disease-free survival in patients with nasopharyngeal carcinoma.

6. The application as described in claim 5, characterized in that, The products mentioned are reagents, kits, test strips, chips, or proteomics analysis products.

7. The application as described in claim 5 or 6, characterized in that, The test sample for the product is blood or its fraction obtained from a self-biopsy subject.

8. The application as described in claim 5 or 6, characterized in that, The test sample for the product was serum.

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