CN112362871B

CN112362871B - Biomarker for esophageal cancer and application thereof

Info

Publication number: CN112362871B
Application number: CN202011130430.7A
Authority: CN
Inventors: 孙苏彭; 周兴宇; 隗啸南; 杨盼盼; 周静; 孙立平
Original assignee: Hangzhou Kaibaoluo Biological Science & Technology Co ltd
Current assignee: Hangzhou Kaibaoluo Biological Science & Technology Co ltd
Priority date: 2020-10-21
Filing date: 2020-10-21
Publication date: 2023-06-06
Anticipated expiration: 2040-10-21
Also published as: CN112362871A; WO2022083673A1

Abstract

The invention provides a biomarker for esophageal cancer, which is an autoantibody combination and comprises at least three autoantibodies respectively resisting the following tumor antigens: trim21, CIP2A, CTAG, CCDC110, mage, MAGEA4, RALA and PDE4DIP. Early screening for esophageal cancer can be achieved by detecting the biomarker. The invention also provides an antigen protein combination for detecting the biomarker, a kit comprising the antigen protein combination, and a corresponding detection or diagnosis method.

Description

Biomarker for esophageal cancer and application thereof

Technical Field

The invention relates to the field of biotechnology and medical diagnosis, in particular to an autoantibody biomarker for esophageal cancer, an antigen combination for detecting the autoantibody biomarker and application of the autoantibody biomarker and the antigen combination in esophageal cancer detection.

Background

Esophageal cancer is a common malignancy worldwide. China is a country with high esophageal cancer incidence, 23 thousands of new cases are increased annually, and the incidence rate is about half of the world, the 5 th place of malignant tumor and the 4 th place of death rate are located, and the incidence rate has a trend of low age; however, the incidence rate of esophageal cancer in developed European and American countries is significantly lower than that in China, but the number of new cases per year is as high as 1.7 ten thousand. High risk factors for esophageal cancer include smoking, drinking, overeating fruits and vegetables, esophageal reflux, obesity, smoking, and the like; age, sex (male) and family history also account for certain risk factors.

Esophageal cancer does not have obvious feeling at the initial stage of disease, and does not draw attention of people; once clinical signs such as dysphagia, poststernal pain or discomfort occur, about 90% of patients are already at mid-late stage and have poor therapeutic effects. Therefore, the method for effectively screening the early lesions of the esophageal cancer has great clinical significance.

Currently, endoscopy-biopsy is mainly used for early detection of esophageal cancer in high-risk groups. However, operator habits are different or biopsy sampling errors may lead to detection errors. Furthermore, the early stage of esophageal endothelial cell tissue proliferation is distributed in a pudding type, and the distribution in biopsy tissues is uneven, so that the difficulty of early diagnosis is increased. At the same time, monitoring proliferation of esophageal epithelial cells is not necessarily effective in early diagnosis of esophageal cancer.

Detection of esophageal cancer using blood markers such as tumor antigen markers, circulating DNA, methylated fragments, or mirnas, etc. has also been proposed. Such markers are usually derived directly from tumor cells, from which they die or actively release signals into the circulatory system. Since early tumors have a small number of cancer cells, only a few of them die and actively release, and these markers have a short half-life in the circulatory system, this signal from the tumor is very weak or even absent and cannot be used as a good marker for early tumors. Meanwhile, due to the fact that the number of detection steps or the standardization of the markers is large, the markers have the defects of being difficult to apply clinically, long in period, high in cost and the like.

Autoantibodies refer to antibodies to self tissues, organs, cells, and cellular components. In early stages of cancer development, exposure of tumor-associated antigens can be recognized by the human immune system, producing tumor-associated autoantibodies, which can be sensitively detected by conventional means in the art, and which can maintain high levels of autoantibodies even in peripheral blood. It is well accepted in the art that autoantibodies generated by tumor antigens are good indicators for early diagnosis of tumors, and that the utilization of autoantibodies generated by tumor induction to reflect the tumor generation mechanism and disease progression of patients is becoming an important direction for finding new targets for early diagnosis and prognosis of tumors.

Autoantibody detection has been proposed for use in esophageal cancer screening. However, due to tumor heterogeneity and differences in immune system response between individuals, the sensitivity of individual tumor autoantibodies in tumor patients is not high enough, typically only 5% -20%. Thus, the combination of a plurality of different tumor autoantibodies can increase the sensitivity of detection.

Disclosure of Invention

The combination of a plurality of autoantibodies for clinical diagnosis of esophageal cancer can obviously improve the accuracy of diagnosing early esophageal cancer, and the earlier the esophageal cancer is found, the greater the clinical treatment significance. Therefore, in order to solve the technical problems, the invention finally identifies a group of autoantibodies which can be used for screening esophageal cancer, in particular early-stage esophageal cancer by detecting the autoantibodies aiming at different tumor antigen targets in the blood of an esophageal cancer patient. The autoantibody combination is used as a biomarker, has high enough sensitivity in especially early tumors, and is especially used in experimental Chinese people; while also having a sufficiently high detection specificity.

It is therefore an object of the present invention to provide a biomarker for esophageal cancer that is an autoantibody combination.

Based on this autoantibody combination as biomarker, it is another object of the invention to provide reagents for detecting this autoantibody combination, such as antigen protein combinations; and provides the application of the autoantibody combination or the detection reagent in preparing products for disease risk prediction, screening, prognosis evaluation, treatment effect monitoring or recurrence monitoring of esophageal cancer and the like.

It is a further object of the invention to provide a kit and a method for the prediction of risk of developing esophageal cancer, screening, prognostic assessment, monitoring of therapeutic effect or monitoring of recurrence, etc. accordingly.

The technical scheme of the invention is as follows.

In one aspect, the invention provides a biomarker for esophageal cancer that is an autoantibody combination comprising at least three of the autoantibodies respectively against the following tumor antigens: trim21, CIP2A, CTAG, CCDC110, mage, MAGEA4, RALA and PDE4DIP.

Preferably, the autoantibody combination comprises autoantibodies against the following tumor antigens, respectively: CCDC110, MAGEA4 and PDE4DIP.

More preferably, the autoantibody combination further comprises at least one, at least two, at least three, at least four or even at least five of the autoantibodies against the following tumor antigens, respectively: CTAG2, trim21, CIP2A, CAGE and RALA. Wherein the autoantibody combination preferably comprises autoantibodies against the following tumor antigens: CTAG2.

Further preferred, the autoantibody combination further comprises an autoantibody against a tumor antigen of: trim21 and RALA; and/or CIP2A and CAGE.

According to a specific embodiment of the invention, the autoantibody combination comprises autoantibodies against the following tumor antigens, respectively:

(1) CCDC110, MAGEA4 and PDE4DIP;

(2) CCDC110, CTAG2, MAGEA4 and PDE4DIP;

(3) CCDC110, CTAG2, MAGEA4, PDE4DIP, trim21, and RALA;

(4) CCDC110, CTAG2, MAGEA4, PDE4DIP, CIP2A, and CAGE;

(5) CCDC110, CTAG2, MAGEA4, PDE4DIP, trim21, RALA, CIP2A, and CAGE.

According to the invention, the autoantibodies are autoantibodies in whole blood, serum, plasma, tissue or cells, interstitial fluid, cerebrospinal fluid or urine of a subject; wherein preferably the tissue or cell is esophageal tissue or cell, esophageal cancer tissue or cell or a paracancerous tissue or cell of esophageal cancer.

Preferably, the subject is a mammal, preferably a primate mammal, more preferably a human. For example, the subject is a chinese population.

Preferably, the autoantibody is IgA (e.g., igA1, igA 2), igM, or IgG (e.g., igG1, igG2, igG3, igG 4).

Preferably, the esophageal cancer includes esophageal squamous carcinoma, low-differentiation carcinoma of esophagus, adenocarcinoma of esophagus, and neuroendocrine carcinoma of esophagus; alternatively, the esophageal cancer comprises stage 0, stage I, stage II, stage III.

According to the invention, the biomarker, i.e. the autoantibody combination, can be detected in a sample (e.g. plasma or serum) of the subject. In the present invention, "presence" or "absence" of autoantibodies is used interchangeably with "positive" or "negative"; this is judged as conventional in the art. For example, detection can be by a tumor-associated antigen and antigen-antibody specific reaction therebetween that results in the presence of any autoantibody in the combination. Accordingly, in a further aspect, the invention also provides a reagent for detecting a biomarker of the invention.

Depending on the specific technical means, the reagents may be reagents for enzyme-linked immunosorbent assay (ELISA), protein/peptide fragment chip detection, immunoblotting, microbead immunoassay, microfluidic immunoassay, or the like. Preferably, the reagents are used to detect the biomarkers of the invention by antigen-antibody reaction, for example by ELISA or fluorescent or chemiluminescent immunoassay.

In this aspect, the agent may be an antigen protein combination comprising at least three of the following tumor antigens: trim21, CIP2A, CTAG, CCDC110, mage, MAGEA4, RALA and PDE4DIP.

Preferably, the antigen protein combination comprises the following tumor antigens: CCDC110, MAGEA4 and PDE4DIP.

More preferably, the antigen protein combination further comprises at least one, at least two, at least three, at least four or even at least five of the following tumor antigens: CTAG2, trim21, CIP2A, CAGE and RALA. Wherein the antigen protein combination preferably comprises the following tumor antigens: CTAG2. Further preferred, the autoantibody combination further comprises an autoantibody against a tumor antigen of: trim21 and RALA; and/or CIP2A and CAGE;

according to a specific embodiment of the invention, the antigen protein combination comprises the following tumor antigens:

(1) CCDC110, MAGEA4 and PDE4DIP;

(2) CCDC110, CTAG2, MAGEA4 and PDE4DIP;

(3) CCDC110, CTAG2, MAGEA4, PDE4DIP, trim21, and RALA;

(4) CCDC110, CTAG2, MAGEA4, PDE4DIP, CIP2A, and CAGE;

(5) CCDC110, CTAG2, MAGEA4, PDE4DIP, trim21, RALA, CIP2A, and CAGE.

In a further aspect, the invention provides the use of the biomarker or agent in the manufacture of a product for the prediction of risk of developing esophageal cancer, screening, prognostic assessment, monitoring of therapeutic effect or monitoring of recurrence.

Preferably, the esophageal cancer includes esophageal squamous carcinoma, low-differentiation carcinoma of esophagus, adenocarcinoma of esophagus, and neuroendocrine carcinoma of esophagus; alternatively, the esophageal cancer includes stage 0, stage I, stage II, stage III esophageal cancer.

Preferably, the product is a kit; more preferably, the kit is a kit for enzyme-linked immunosorbent assay (ELISA), protein/peptide fragment chip detection, immunoblotting, microbead immunoassay or microfluidic immunoassay. Preferably, the kit is for detecting the biomarker by antigen-antibody reaction, for example an ELISA kit or a fluorescent or chemiluminescent immunoassay kit.

In yet another aspect, the invention provides a kit comprising the agent of the invention.

Depending on the specific technical means, the kit may be a kit for enzyme-linked immunosorbent assay (ELISA), protein/peptide fragment chip detection, immunoblotting, microbead immunoassay, microfluidic immunoassay, or the like. Preferably, the kit is used for detection of the biomarker of the invention by antigen-antibody reaction, for example an ELISA kit or a fluorescent or chemiluminescent immunoassay kit.

Thus, preferably, the kit is an enzyme-linked immunosorbent assay (ELISA) detection kit. That is, the kit is used to detect whether or not the autoantibody biomarker in the sample of the subject is positive by the enzyme-linked immunosorbent assay. Accordingly, the kit may also include other components required for ELISA detection of autoantibody biomarkers, all as is well known in the art. For detection purposes, for example, the antigen protein in the kit may be linked to a tag peptide, such as His tag, streptavidin tag, myc tag; for another example, the kit may include a solid support, such as a support having microwells to which antigen proteins can be immobilized, such as an elisa plate; or a microbead or magnetic bead solid phase carrier. It may also include an adsorption protein for immobilizing an antigen protein on a solid carrier, a dilution of blood such as serum, a washing solution, a secondary antibody with an enzyme label or a fluorescent or chemiluminescent substance, a color development solution, a stop solution, etc. The concentration of the corresponding antibody in the body fluid is detected by the principle that the antigen protein indirectly or directly coated on the surface of the solid carrier reacts with the antibody in serum/plasma/tissue fluid and the like to form an antigen-antibody complex.

In yet another aspect, the present invention provides a method for disease risk prediction, screening, prognosis evaluation, treatment effect monitoring or recurrence monitoring of esophageal cancer, comprising the steps of:

(1) Quantifying each autoantibody in the autoantibody combination provided by the invention in a sample from a subject;

(2) Comparing the amount of the autoantibody to a reference threshold, and determining that the subject is at risk of having, has a poor prognosis of, or has a poor treatment for esophageal cancer when it is above the reference threshold.

In step (1), the quantification comprises detection of each autoantibody in the autoantibody combination using the reagent (i.e. antigen protein combination) or the kit comprising the reagent provided by the invention.

According to the invention, the subject is a mammal, preferably a primate mammal, more preferably a human. And, preferably, the esophageal cancer includes esophageal squamous carcinoma, low-differentiation carcinoma of esophagus, adenocarcinoma of esophagus, and neuroendocrine carcinoma of esophagus; alternatively, the esophageal cancer includes stage 0, stage I, stage II, stage III esophageal cancer.

According to the invention, the sample is whole blood, serum, plasma, tissue or cells, interstitial fluid, cerebrospinal fluid or urine of a subject; wherein preferably the tissue or cell is esophageal tissue or cell, esophageal cancer tissue or cell or a paracancerous tissue or cell of esophageal cancer.

In step (2), the reference threshold may be a reference level from a healthy person or population of healthy people; for example, it may be defined as the mean value of a population confirmed by physical examination as not having cancer plus 2 standard deviations or 3 standard deviations.

Compared with the prior art, the invention provides a novel biomarker for esophageal cancer, which is a brand-new group of tumor autoantibodies. Each of these autoantibodies has a separate positive contribution to the detection of esophageal cancer, and has a fairly high detection specificity and sensitivity. When used in combination, the kit has quite high detection sensitivity and detection specificity, and can reach 50% of sensitivity even in the case of stage 0 esophageal cancer.

Drawings

Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a scatter plot of the horizontal distribution of each tumor autoantibody in tumor and control groups.

Detailed Description

In the present invention, the term "antigen" or the term "antigenic protein" is used interchangeably. Furthermore, the present invention relates to the following experimental operations or definitions. It should be noted that the present invention may also be practiced using other techniques conventional in the art and is not limited to the following experimental procedures.

Preparation of recombinant antigen proteins

The cDNA fragment of the tumor antigen was cloned into PET28 (a) expression vector containing the 6XHIS tag. At the N-or C-terminus of the antigen, streptavidin or the like (biotin-binding tag protein) is introduced. The obtained recombinant expression vector is transformed into escherichia coli for expression. The protein expressed by the supernatant was purified by Ni-NTA affinity column and ion column. When the protein is expressed in inclusion bodies, the protein is denatured by 6M guanidine hydrochloride, renaturated and folded in vitro according to a standard method, and then purified by a Ni-NTA affinity column through a 6XHIS tag, so that antigen protein is obtained.

(II) preparation and preservation of serum or plasma

Serum or plasma from patients with esophageal cancer is collected when the patients are initially diagnosed with esophageal cancer and have not received any radiotherapy or chemotherapy or surgical treatment. Plasma or serum was prepared according to standard clinical procedures and stored in a-80 ℃ refrigerator for long periods of time.

(III) ELISA detection

The concentration of autoantibodies in the sample was quantified by enzyme-linked immunosorbent assay (ELISA).

The purified tumor antigen is immobilized to the microwell surface by its tag streptavidin or the like. Microwells were pre-coated with biotin-labeled Bovine Serum Albumin (BSA). Serum or plasma samples were diluted 1:110 fold with phosphate buffer and reacted by adding microwells (50 ml/well). After washing unbound serum or plasma components with wash solution, horseradish peroxidase (HRP) -conjugated anti-human IgG was added to each well for reaction. Then, TMB (3, 3', 5' -tetramethylbenzidine) as a reaction substrate was added for color development. Stop solution (1N HCl) was added and absorbance was measured at 450nm using a microplate reader (OD). Serum autoantibody concentrations were quantified using a standard curve.

Critical value (cutoff value) of autoantibody

The cutoff value of an autoantibody is defined as the mean value of the detected absorbance values plus 2 Standard Deviations (SD) or the mean value plus 3 Standard Deviations (SD) in a control normal population confirmed by physical examination to have no cancer. Determination of the cutoff value for each autoantibody was based on the following principle: 1. in the case of using two different values (average plus two standard deviations and average plus three standard deviations) as reference thresholds, the detection specificity of each autoantibody to the control normal population is 95% or more; 2. and under the condition that the two different values are adopted as reference thresholds, obtaining the specificity of each autoantibody to the control normal population and the sensitivity of each autoantibody to the population of patients with esophageal cancer, calculating the sum of the specificity and the sensitivity, and selecting the value with the sum being larger as the determined cutoff value of the autoantibody.

(V) determination of the positivity and negativity of an individual autoantibody

For each autoantibody assay, positive response is defined as quantifying the level of autoantibody in the sample, and then comparing it with the cutoff value, which is not less than the cutoff value positive; accordingly, a negative response is defined as < cutoff value negative.

Positive determination of autoantibody combinations

Since the single autoantibody has a low positive rate, the result is analyzed by combining the results of a plurality of autoantibodies to determine the predictive effect in order to increase the positive rate of autoantibody detection. The rules are: detecting a plurality of autoantibodies in a sample, and judging that the combined result of the antibodies is positive as long as one or more autoantibodies show positive; if all the autoantibodies are negative, the antibody combination result is judged to be negative.

(seventh) statistical analysis method

Both groups were statistically analyzed using GraphPad Prism v.6 (GraphPad Prism software, san diego, california) and IBM SPSS Statistics (IBM, new york) for Windows using the Mann-Whitney U test. In analyzing the relationship between each parameter, a Spearman correlation analysis was performed.

Eighth sensitivity and specificity determination

Sensitivity: in all cases of esophageal cancer diagnosed by gold standard, the cases with positive autoantibodies or autoantibody combination detection results account for the proportion of all cases with diseases.

Specificity: among all subjects diagnosed with no disease by gold standard, the subjects whose autoantibodies or autoantibody combination detection results were negative are present in proportion to all subjects with no disease.

The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way. Sample collection has informed consent of the subject or patient and is approved by regulatory authorities.

The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials and the like used in the examples described below are commercially available products unless otherwise specified.

Example 1

Screening for tumor autoantibody markers was performed using a discovery cohort, including 47 healthy physical examination populations and 41 esophageal cancer patients. Serum samples of patients with esophageal cancer come from Shanghai tumor Hospital, and serum samples of healthy physical examination population come from Shanghai tumor Hospital and physical examination center.

All the serum of the patients with the esophageal cancer is collected when the patients are initially diagnosed with the esophageal cancer and not subjected to any radiotherapy and surgery treatment, and is stored in a refrigerator of-80 ℃.

Patient information used for the discovery queue is shown in table 1.

Table 1: discovery queue patient feature table

17 esophageal cancer related antigens are selected, expressed and purified, coated on the surface of a 96-well plate, sealed, reacted with serum of esophageal cancer patient population diluted 1:110 times or serum of normal control population, then reacted with anti-human IgG antibody of HRP horseradish peroxidase, then subjected to chromogenic reaction, and detected by an enzyme-labeled instrument OD450nm wavelength. Table 2 shows the sensitivity and specificity of autoantibodies corresponding to each antigen.

Table 2: sensitivity and specificity of single tumor autoantibody markers of the discovery cohort

Antigens	Sequence number	Sensitivity to	Specificity (specificity)
				Trim21	NM_003141.4	15％	98％
CIP2A	NM_020890.3	7％	100％
				CTAG2	NM_020994.5	15％	100％
CCDC110	NM_152775.4	27％	100％
				CAGE	NM_182699.4	17％	100％
RAL-A	NM_005402.4	7％	96％
				PDE4DIP	NM_001002811.2	12％	100％
MAGEA4	NM_001011548.1	22％	96％
				SURF1	NM_003172.4	8％	98％
GLUT1	NM_006516.4	6％	100％
				BORIS	NM_001269040.2	12％	98％
HSP105	NM_006644.4	8％	100％
				Annexin 1	NM_000700.3	6％	100％
SOX2	NM_003106.4	12％	96％
				PRDX1	NM_002574.4	8％	98％
BRCA1(1-110)	NM_007294.4	22％	98％
				TROP2	NM_002353.3	10％	100％

Autoantibodies corresponding to the 17 antigens were subjected to three-layer sorting as follows:

1. the inventor finds in the previous study that the comparison crowd has larger bias due to physical examination from physical examination crowd samples of different areas or hospitals, so that the comparison crowd using a plurality of different places or hospitals can more accurately represent the actual scene of clinical application, thereby making the detection have more clinical significance. Thus, in this study, 90 new healthy control populations were used to perform serum detection of the tumor antigen autoantibodies, and antigens with specificity of 94% or less in the new control serum, such as BRCA1 (1-110) and BORIS, were removed.

2. Some antigens have higher sensitivity, but because of positive detection overlapped with other antigens, there is no single positive contribution rate, so that some antigens such as HSP105, annexin1, SOX2, PRDX1, TROP2 and the like are removed.

By way of comprehensive consideration, a preferred set of autoantibody combinations is selected, comprising autoantibodies against the following 8 tumor associated antigens, respectively: trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4DIP. Serum testing of these 8 autoantibodies in the discovery cohort using the corresponding 8 tumor antigens found sensitivity and specificity of the combination of these 8 autoantibodies to be 70.7% and 87.0%, respectively (table 3); whereas the detection sensitivity at the T0, T1, T2, T3 phases was 50%,88%,63% and 50%, respectively, as analyzed according to the esophageal carcinoma stage (Table 4).

Table 3: detection results of discovery of queued tumor autoantibody combinations

Number of people	Number of positives	Sensitivity to	Specificity (specificity)
				41 (patient)	29	70.7％
47 (health)	6		87.0％

Table 4: detection sensitivity of different stages of combination of tumor autoantibodies in queue was found

Stage by stage	Number of patients	Number of positives	Sensitivity (%)
				T0	4	2	50
T1	8	7	88
				T2	8	5	63
T3	6	3	50
				Unknown	15	12	80

Example 2

The validation cohort included 129 esophageal cancer patients freshly collected from Shanghai tumor hospitals (118 cases cancer, 6 cases poorly differentiated cancer, 2 cases adenocarcinoma, 3 cases neuroendocrine cancer), and 88 physical examination populations collected from Shanghai tumor hospitals and physical examination centers for validation of antibody markers. Patient information is shown in table 5.

Table 5: verification queue patient feature table

The horizontal distribution of each tumor autoantibody in the tumor group and the control group was shown by scatter plots for antigens Trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4DIP (see fig. 1). Statistical analysis of the antibody level distribution of tumor autoantibodies in tumor and control groups using Mann Whitney test found that the level distribution of autoantibodies against tumor antigens CTAG2, trim21 and RALA was significantly different in tumor and control groups (p < 0.05); whereas the level distribution of autoantibodies against the tumor antigens CIP2A, CCDC110, mage, MAGEA4 and PDE4DIP was not significantly different in the tumor group and the control group. However, if the levels of autoantibody markers greater than the cutoff value in the tumor and control groups were analyzed to be distributed between the two groups of people, they were very different. Because of this, all these positive markers greater than the cutoff value are of great significance for highly specific detection of patients with esophageal cancer tumors.

The sensitivity and specificity of detection of 8 tumor autoantibody markers in 129 esophageal cancer sera and 88 healthy control sera of the validation cohort are shown in table 6. The specificity of the single marker is 96.6% or above, and the sensitivity is 7.0-17.1%.

Table 6: validation of detection sensitivity and specificity of individual autoantibodies of the cohort

	Sensitivity (%)	Specificity (%)
			CAGE	16.3	98.9
CCDC110	9.3	98.8
			CIP2A	7.0	97.7
CTAG2	17.1	100.0
			MAGEA4	7.8	98.8
TRIM21	14.0	96.6
			RALA	8.5	98.8
PDE4DIP	7.8	98.8

Different antigens were selected from the 8 antigens to form different antigen combinations, from which detection of the corresponding tumor autoantibody combinations was performed, and the results are shown in table 7. It can be seen that as the number of antigens increases, the detection sensitivity increases and the specificity decreases gradually. The three autoantibody combinations (anti-CCDC 110, MAGEA4 and PDE4 DIP) detection sensitivity and specificity were 41.5% and 93.6%, respectively, and had reached quite high levels. One autoantibody, such as anti-CTAG 2, is added on the basis of the combination of the three autoantibodies, the detection sensitivity is increased to 53.6%, and the specificity is kept unchanged. Further, the increased sensitivity of the formed autoantibody combinations is higher, while the specificity remains around 90% or even higher, continuing to increase the different autoantibody markers. For example, increasing the sensitivity and specificity of Trim21 and RALA assays on this basis was 65.8% and 89.3%, respectively; whereas the sensitivity and specificity of the autoantibody marker combinations comprising against 8 tumor antigens Trim21, CIP2A, CTAG, CCDC110, CAGE, MAGEA4, RALA and PDE4DIP were 70.7% and 87.2%, respectively, in the validation cohort.

Table 7: verifying the sensitivity and specificity of combinations of different tumor antigens in a cohort to detect autoantibody markers

Example 3

Autoantibody marker combinations against 8 tumor antigens Trim21, CIP2A, CTAG2, CCDC110, mage, MAGEA4, RALA and PDE4DIP were selected for susceptibility analysis of different stages and types of esophageal cancer.

Referring to example 2, the validation cohort included 129 esophageal cancer patients (118 cases cancer, 6 cases poorly differentiated cancer, 2 cases adenocarcinoma, 3 cases neuroendocrine cancer). Verification cohort 129 esophageal cancer patients included 3T 0 phases, 25T 1 phases, with sensitivity of detection of 0% and 32%, respectively; the sensitivities were 72% and 60% for the 18T 2 phases and 58T 3 phases, respectively; the remaining 25 cases had no information on the phase, and the sensitivity was 60%. The results are shown in Table 8.

Table 8: verifying sensitivity of different stages of the queue autoantibody combination

Most of the validation queues were carcinomas, 118. Although there is a decrease in the incidence of squamous carcinoma in esophageal cancer in western developed countries, there is an increase in the incidence of adenocarcinoma, most of those currently in China are carcinoma. As shown by the detection results, the detection sensitivity of the preferred combination of 8 autoantibodies of the invention to squamous cell carcinoma reaches 53%. In addition, the validation cohort included 2 adenocarcinomas, 3 neuroendocrine carcinomas, and the positive detection rate of the autoantibody combinations were all 100%. There are also 6 cases of poorly differentiated cancers, whose detection sensitivity is 67%. The results are shown in Table 9.

Table 9: verifying sensitivity of different subtypes of a combination of queue autoantibodies

Tumor subtype	Number of patients	Sensitivity (%)
			Adenocarcinoma of gland	2	100
Neuroendocrine cancer	3	100
			Hypodifferentiation cancer	6	67
cancer	118	53

The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications according to the present invention without departing from the spirit of the present invention, and shall fall within the scope of the appended claims.

Claims

1. Use of a biomarker, or an agent for detecting the biomarker, in the manufacture of a product for screening for esophageal cancer, the biomarker being an autoantibody combination comprising autoantibodies against the following tumor antigens, respectively: CCDC110, MAGEA4 and PDE4DIP.

2. The use according to claim 1, wherein the screening is an early screening.

3. The use according to claim 1, wherein the autoantibody combination further comprises at least one, at least two, at least three, at least four or even at least five of the autoantibodies against the following tumour antigens, respectively: CTAG2, trim21, CIP2A, CAGE and RALA.

4. The use according to claim 1, wherein the autoantibody combination comprises autoantibodies against the following tumour antigens: CTAG2.

5. The use according to claim 4, wherein the autoantibody combination further comprises autoantibodies against the following tumour antigens: trim21 and RALA; and/or CIP2A and CAGE.

6. The use according to claim 1, wherein the autoantibody combination comprises autoantibodies against the following tumour antigens respectively:

CCDC110, CTAG2, MAGEA4 and PDE4DIP;

CCDC110, CTAG2, MAGEA4, PDE4DIP, trim21, and RALA;

CCDC110, CTAG2, MAGEA4, PDE4DIP, CIP2A, and CAGE; or (b)

CCDC110, CTAG2, MAGEA4, PDE4DIP, trim21, RALA, CIP2A, and CAGE.

7. The use according to any one of claims 1 to 6, wherein the autoantibody is an autoantibody in whole blood, serum, plasma, tissue or cells, interstitial fluid, cerebrospinal fluid or urine of a subject.

8. The use according to claim 7, wherein the tissue or cell is esophageal tissue or cell, esophageal cancer tissue or cell or a paracancerous tissue or cell of esophageal cancer.

9. The use of claim 7, wherein the subject is a mammal.

10. The use of claim 7, wherein the subject is a primate mammal.

11. The use of claim 7, wherein the subject is a human.

12. The use according to any one of claims 1 to 6, wherein the autoantibody is IgA, igM or IgG.

13. The use according to any one of claims 1 to 6, wherein the esophageal cancer comprises esophageal squamous carcinoma, poorly differentiated carcinoma of the esophagus, adenocarcinoma of the esophagus, and neuroendocrine carcinoma of the esophagus; alternatively, the esophageal cancer comprises stage 0, stage I, stage II, stage III.

14. The use according to any one of claims 1 to 6, wherein the reagent is a reagent for enzyme-linked immunosorbent assay, protein/peptide fragment chip detection, immunoblotting, microbead immunoassay or microfluidic immunoassay.

15. The use according to any one of claims 1 to 6, wherein the reagent is for detection of the biomarker by an antigen-antibody reaction.

16. The use according to any one of claims 1 to 6, wherein the reagent is used for detection of the biomarker by enzyme-linked immunosorbent assay or fluorescent or chemiluminescent immunity.

17. The use according to any one of claims 1 to 6, wherein the agent is an antigen protein combination comprising the following tumor antigens: CCDC110, MAGEA4 and PDE4DIP.

18. The use according to claim 17, wherein the antigen protein combination further comprises at least one, at least two, at least three, at least four or even at least five of the following tumor antigens: CTAG2, trim21, CIP2A, CAGE and RALA.

19. The use according to claim 17, wherein the antigen protein combination comprises the following tumor antigens: CTAG2.

20. The use according to claim 19, wherein the antigen protein combination further comprises the following tumor antigens: trim21 and RALA; and/or CIP2A and CAGE.

21. The use according to claim 17, wherein the antigen protein combination comprises the following tumor antigens:

CCDC110, CTAG2, MAGEA4 and PDE4DIP;

CCDC110, CTAG2, MAGEA4, PDE4DIP, trim21, and RALA;

CCDC110, CTAG2, MAGEA4, PDE4DIP, CIP2A, and CAGE; or (b)

CCDC110, CTAG2, MAGEA4, PDE4DIP, trim21, RALA, CIP2A, and CAGE.

22. The use according to claim 1, wherein the product is a kit.

23. The use according to claim 22, wherein the kit is a kit for enzyme-linked immunosorbent assay, protein/peptide fragment chip detection, immunoblotting, microbead immunoassay or microfluidic immunoassay.

24. The use according to claim 22, wherein the kit is for detecting the biomarker by an antigen-antibody reaction.

25. The use according to claim 22, wherein the kit is an enzyme-linked immunosorbent assay kit or a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

26. A kit comprising reagents for detecting a biomarker as defined in any of claims 1 to 13.

27. The kit of claim 26, wherein the reagent is a reagent for enzyme-linked immunosorbent assay, protein/peptide fragment chip detection, immunoblotting, microbead immunoassay, or microfluidic immunoassay.

28. The kit of claim 26, wherein the reagents are for detecting the biomarker by an antigen-antibody reaction.

29. The kit of claim 26, wherein the reagents are for detecting the biomarker by enzyme-linked immunosorbent assay or fluorescent or chemiluminescent immunoassay.

30. The kit of claim 26, wherein the agent is an antigen protein combination comprising the following tumor antigens: CCDC110, MAGEA4 and PDE4DIP.

31. The kit of claim 30, wherein the antigen protein combination further comprises at least one, at least two, at least three, at least four, or even at least five of the following tumor antigens: CTAG2, trim21, CIP2A, CAGE and RALA.

32. The kit of claim 30, wherein the antigen protein combination comprises the following tumor antigens: CTAG2.

33. The kit of claim 32, wherein the antigen protein combination further comprises the following tumor antigens: trim21 and RALA; and/or CIP2A and CAGE.

34. The kit of claim 30, wherein the antigen protein combination comprises the following tumor antigens:

CCDC110, CTAG2, MAGEA4 and PDE4DIP;

CCDC110, CTAG2, MAGEA4, PDE4DIP, trim21, and RALA;

CCDC110, CTAG2, MAGEA4, PDE4DIP, CIP2A, and CAGE; or (b)

CCDC110, CTAG2, MAGEA4, PDE4DIP, trim21, RALA, CIP2A, and CAGE.

35. The kit of any one of claims 26 to 34, wherein the kit is a kit for enzyme-linked immunosorbent assay, protein/peptide fragment chip detection, immunoblotting, microbead immunoassay or microfluidic immunoassay.

36. The kit of any one of claims 26 to 34, wherein the kit is for detection of the biomarker by an antigen-antibody reaction.

37. The kit of any one of claims 26 to 34, wherein the kit is an enzyme-linked immunosorbent assay kit or a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.