CN112362871A - Biomarkers of esophageal cancer and application thereof - Google Patents
Biomarkers of esophageal cancer and application thereof Download PDFInfo
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Abstract
The invention provides a biomarker of esophageal cancer, which is an autoantibody combination comprising at least three autoantibodies against the following tumor antigens respectively: trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4 DIP. Early screening for esophageal cancer can be achieved by detecting the biomarkers. The invention also provides an antigen protein combination for detecting the biomarker and a kit containing the antigen protein combination, and a corresponding detection or diagnosis method.
Description
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 in esophageal cancer detection.
Background
Esophageal cancer is a common malignant tumor worldwide. China is a country with high incidence of esophageal cancer, 23 thousands of new cases are generated every year, the new cases account for about half of the world, the incidence rate is 5 th of malignant tumors, the mortality rate is 4 th, and the incidence rate has a low-age trend; the incidence rate of esophageal cancer in developed countries in Europe and America is obviously lower than that in China, but 1.7 ten thousand of new cases are generated every year. High risk factors for esophageal cancer include smoking, alcohol consumption, too little eating of fruits and vegetables, esophageal reflux, obesity, smoking, and the like; age, sex (male) and family history also account for certain risk factors.
The esophagus cancer has no obvious feeling at the early stage of the onset and cannot attract the attention of people; once clinical signs such as dysphagia, post-sternal pain or discomfort have occurred, about 90% of patients are already in the middle and advanced stage and the treatment is poor. Therefore, the method has great clinical significance for effectively screening early-stage lesion of the esophageal cancer.
Currently, endoscopy-biopsy is mainly used for early detection of esophageal cancer in high risk groups. However, the habit of the operator is different or the biopsy sampling is wrong, which may cause the detection error. Furthermore, the esophageal endothelial cell tissue hyperplasia is distributed in a pudding manner in the early stage, and is unevenly distributed in the biopsy tissue, so that the difficulty of early diagnosis is increased. At the same time, monitoring the proliferation of esophageal epithelial cells is not necessarily effective for early diagnosis of esophageal cancer.
The detection of esophageal cancer using blood markers such as tumor antigen markers, circulating DNA, methylated fragments, or miRNA has also been proposed. Such markers are usually derived directly from tumor cells, from the tumor into the circulatory system due to tumor cell death or active release of a signal. Because of the small number of cancer cells in early stage tumors, only a few of them die and are actively released, and the short half-life of these markers in the circulation, the signals from tumors are very weak or even none, and thus they cannot be used as good markers for early stage tumors. Meanwhile, the detection steps of the markers are multiple or the standardization is insufficient, so that the clinical application has the defects of high difficulty, long period, high cost and the like.
Autoantibodies refer to antibodies directed against self tissues, organs, cells and cellular components. In the early stages of carcinogenesis, exposure to tumor-associated antigens can be recognized by the human immune system to produce tumor-associated autoantibodies, which can be sensitively detected by means of conventional techniques in the art, and can maintain high levels of autoantibodies even in peripheral blood. The field admittedly recognizes that the autoantibody generated by tumor antigen is a better index for early diagnosis of tumor, and the autoantibody generated by tumor induction is used to reflect the tumor generating mechanism and the disease progression process of patients, and is becoming an important direction for searching new targets for early diagnosis and prognosis of tumor.
Autoantibody detection has been proposed for use in esophageal cancer screening. However, due to tumor heterogeneity and differences in immune system response between different individuals, the sensitivity of individual tumor autoantibodies in neoplastic patients is not high enough, usually only 5% to 20%. Thus, the combination of multiple different tumor autoantibodies can increase detection sensitivity.
Disclosure of Invention
The combination of several autoantibodies for clinical diagnosis of esophageal cancer can significantly improve the accuracy of early esophageal cancer diagnosis, and the earlier esophageal cancer is found, the greater the clinical therapeutic significance is. Therefore, in order to solve the technical problems, the invention finally identifies a group of autoantibodies for screening esophageal cancer, particularly early esophageal cancer, by detecting the autoantibodies aiming at different tumor antigen targets in the blood of patients with esophageal cancer. The autoantibody combination is used as a biomarker with high enough sensitivity in particularly early tumors, especially in experimental Chinese population; and also has sufficiently high detection specificity.
It is therefore an object of the present invention to provide a biomarker for esophageal cancer which is an autoantibody combination.
Based on the combination of autoantibodies as biomarkers, another object of the invention is to provide reagents for detecting the combination of autoantibodies, such as antigen protein combinations; and provides the application of the autoantibody combination or the detection reagent in preparing products for esophageal cancer risk prediction, screening, prognosis evaluation, treatment effect monitoring or relapse monitoring and the like.
It is still another object of the present invention to provide a kit and a method for predicting the risk of esophageal cancer, screening, prognosis evaluation, monitoring the effect of treatment, monitoring recurrence, etc., accordingly.
The technical scheme of the invention is as follows.
In one aspect, the invention provides a biomarker for esophageal cancer, the biomarker being an autoantibody combination comprising at least three of the autoantibodies against the following tumor antigens respectively: trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4 DIP.
Preferably, the combination of autoantibodies comprises autoantibodies against the following tumor antigens respectively: CCDC110, MAGEA4, and PDE4 DIP.
More preferably, the combination of autoantibodies 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 combination of autoantibodies preferably comprises autoantibodies against the following tumor antigens: CTAG 2.
Further preferably, the combination of autoantibodies further comprises autoantibodies against the following tumor antigens: trim21 and RALA; and/or, CIP2A and CAGE.
According to a particular embodiment of the invention, said combination of autoantibodies comprises autoantibodies against the following tumor antigens, respectively:
(1) CCDC110, MAGEA4, and PDE4 DIP;
(2) CCDC110, CTAG2, MAGEA4, and PDE4 DIP;
(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 in whole blood, serum, plasma, tissue or cells, interstitial fluid, cerebrospinal fluid or urine of the subject; wherein preferably, the tissue or cell is esophageal tissue or cell, esophageal cancer tissue or cell, or a tissue or cell beside 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, IgA2), IgM or IgG (e.g. IgG1, IgG2, IgG3, IgG 4).
Preferably, the esophageal cancer comprises esophageal squamous carcinoma, esophageal low-differentiation cancer, esophageal adenocarcinoma, and esophageal neuroendocrine cancer; alternatively, the esophageal cancer comprises stage 0, stage I, stage II, and stage III.
According to the present invention, the biomarker, i.e., the combination of autoantibodies, may be detected in a sample (e.g., plasma or serum) from the subject. In the present invention, "presence" or "absence" of autoantibodies is used interchangeably with "positive" or "negative"; judging this is routine in the art. For example, detection can be by an antigen-antibody specific reaction between the tumor associated antigen and any autoantibodies that result in the presence of the combination. Accordingly, in another aspect, the invention also provides a reagent for detecting a biomarker of the invention.
Depending on the specific technical means, the reagent may be a reagent for enzyme-linked immunosorbent assay (ELISA), protein/peptide 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 can be an antigenic protein combination that includes at least three of the following tumor antigens: trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4 DIP.
Preferably, the antigenic protein combination comprises the following tumor antigens: CCDC110, MAGEA4, and PDE4 DIP.
More preferably, the antigenic 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 said antigenic protein combination preferably comprises the following tumor antigens: CTAG 2. Further preferably, the combination of autoantibodies further comprises autoantibodies against the following tumor antigens: trim21 and RALA; and/or, CIP2A and CAGE;
according to a particular embodiment of the invention, said antigenic protein combination comprises the following tumor antigens:
(1) CCDC110, MAGEA4, and PDE4 DIP;
(2) CCDC110, CTAG2, MAGEA4, and PDE4 DIP;
(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 reagent in the manufacture of a product for the prediction of risk of developing esophageal cancer, screening, prognostic assessment, monitoring of therapeutic efficacy or monitoring of recurrence.
Preferably, the esophageal cancer comprises esophageal squamous carcinoma, esophageal low-differentiation cancer, esophageal adenocarcinoma, and esophageal neuroendocrine cancer; alternatively, the esophageal cancer comprises stage 0, stage I, stage II, and 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 immunodetection or microfluidic immunodetection. Preferably, the kit is used for detecting the biomarkers by antigen-antibody reaction, for example, an ELISA kit or a fluorescent or chemiluminescent immunoassay kit.
In yet another aspect, the present invention provides a kit comprising the reagents of the present invention.
Depending on the specific technical means, the kit may be a kit for enzyme-linked immunosorbent assay (ELISA), protein/peptide chip detection, immunoblotting, microbead immunoassay, microfluidic immunoassay, or the like. Preferably, the kit is used for detecting the biomarkers of the invention by antigen-antibody reaction, for example, an ELISA kit or a fluorescent or chemiluminescent immunoassay kit.
Therefore, preferably, the kit is an enzyme-linked immunosorbent assay (ELISA) detection kit. That is, with the kit, whether an autoantibody biomarker in a sample of a subject is positive is detected by an enzyme-linked immunosorbent assay. Accordingly, the kit may also include other components necessary for the ELISA detection of autoantibody biomarkers, all as is well known in the art. For detection purposes, for example, the antigenic protein in the kit may be linked to a tag peptide, such as a His tag, a streptavidin tag, a Myc tag; as another example, the kit may include a solid support, such as a support having a microwell capable of immobilizing an antigen protein, such as an ELISA plate; or a microbead or magnetic bead solid phase carrier. It may further include an adsorption protein for immobilizing an antigen protein on a solid phase carrier, a diluent for blood such as serum, a washing solution, a secondary antibody with an enzyme label or a fluorescent or chemiluminescent substance, a color developing solution, a stop solution, and the like. 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 phase 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 predicting the risk of developing esophageal cancer, screening, prognostically assessing, monitoring the efficacy of a treatment, or monitoring the recurrence of esophageal cancer, comprising the steps of:
(1) quantifying each autoantibody in the combination of autoantibodies provided by the invention in a sample from a subject;
(2) comparing the amount of the autoantibody to a reference threshold and, if it is above the reference threshold, determining that the subject is at risk of, has a poor prognosis or is poorly treated for esophageal cancer.
In step (1), the quantification includes detecting each autoantibody in the autoantibody combination by using the reagent (i.e., antigen-protein combination) provided by the present invention or a kit containing the reagent.
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, esophageal poorly differentiated carcinoma, esophageal adenocarcinoma, and esophageal neuroendocrine carcinoma; alternatively, the esophageal cancer comprises stage 0, stage I, stage II, and 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 the subject; wherein preferably, the tissue or cell is esophageal tissue or cell, esophageal cancer tissue or cell, or a tissue or cell beside esophageal cancer.
In step (2), the reference threshold may be a reference level from a healthy person or a healthy population; for example, it can be defined as the mean plus 2 standard deviations or 3 standard deviations of a population confirmed to have no cancer by physical examination.
Compared with the prior art, the invention provides a novel biomarker of esophageal cancer, which is a brand-new group of tumor autoantibodies. Each of these autoantibodies has an individual positive contribution rate to the detection of esophageal cancer, and has a rather high detection specificity and sensitivity. When used in combination, has quite high detection sensitivity and detection specificity, and can reach 50 percent of sensitivity even in the case of the esophageal cancer of stage 0.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a scattergram of the horizontal distribution of each tumor autoantibody in the tumor group and the control group.
Detailed Description
In the present invention, the term "antigen" or the term "antigenic protein" are used interchangeably. In addition, the following experimental procedures or definitions are involved in the present invention. It should be noted that the present invention can also be implemented by other conventional techniques in the art, and is not limited to the following experimental procedures.
(I) preparation of recombinant antigenic protein
The cDNA fragment of the tumor antigen was cloned into the 6 XHis-tagged PET28(a) expression vector. At the N-or C-terminus of the antigen, streptavidin or an analogue (biotin-binding tag protein) is introduced. The obtained recombinant expression vector is transformed into escherichia coli for expression. The protein expressed from the supernatant was purified by Ni-NTA affinity column and ion column. When the protein is expressed in an inclusion body, the protein is denatured by 6M guanidine hydrochloride, renatured and folded in vitro according to a standard method, and then purified by a Ni-NTA affinity column through a 6XHis tag to obtain the antigen protein.
(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 chemoradiotherapy or surgical treatment. Plasma or serum is prepared according to standard clinical procedures and stored in a refrigerator at-80 deg.C for a long period 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 surface of the microwell by its tag streptavidin or an analogue. The microwells were pre-coated with biotin-labeled Bovine Serum Albumin (BSA). Serum or plasma samples were diluted 1:110 fold with phosphate buffer and added to microwells for reaction (50 ml/well). After washing unbound serum or plasma fractions with a wash solution, horseradish peroxidase (HRP) -conjugated anti-human IgG was added to each well for reaction. Then, a reaction substrate TMB (3,3',5,5' -tetramethylbenzidine) was added to develop color. The stop solution (1N HCl) was added and the absorbance at 450nm was read by an enzyme reader (OD). Serum autoantibody concentrations were quantified using a standard curve.
(IV) cutoff value of autoantibody (cutoff value)
The cutoff value of autoantibodies is defined as the mean plus 2 Standard Deviations (SD) or the mean plus 3 Standard Deviations (SD) of the detected absorbance values in control normal populations confirmed to be free of cancer by physical examination. The cutoff value of each autoantibody was determined according to the following principle: 1. the detection specificity of each autoantibody to the control normal population is 95% or more, using two different values (mean plus two standard deviations and mean plus three standard deviations) as reference thresholds; obtaining the specificity of each autoantibody to the control normal population and the sensitivity to the esophageal cancer patient population by using the two different values as reference thresholds, calculating the sum of the specificity and the sensitivity, and selecting the value in the case of the sum larger as the determined cutoff value of the autoantibody.
(V) Positive and negative judgment of Individual autoantibodies
For each autoantibody assay, a positive reaction is defined as the quantification of the level of autoantibody in the sample, which is then compared to the cutoff value, which is positive for a value of not less than cutoff; accordingly, a negative reaction is defined as a < cutoff value as negative.
(VI) Positive judgment of autoantibody combination
Since the positive rate of a single autoantibody is low, the results are analyzed in combination with the results of a plurality of autoantibodies to determine the prediction effect, in order to increase the positive rate of autoantibody detection. The rule is: detecting a plurality of autoantibodies in the sample, and judging that the antibody combination result is positive if one or more of the autoantibodies is positive; and if all the autoantibodies are negative, judging the antibody combination result as negative.
(VII) statistical analysis method
Both groups were statistically analyzed using the Mann-Whitney U test using Graphpad Prism v.6(Graphpad Prism software, san Diego, Calif.) and IBM SPSS Statistics 23 for Windows (IBM, New York). In analyzing the relationship between each parameter, Spearman's correlation analysis was performed.
(VIII) determination of sensitivity and specificity
Sensitivity: in all cases of esophageal cancer, the proportion of cases with positive detection results of autoantibodies or autoantibody combinations in all cases with pathological conditions is determined by the gold standard.
Specificity: in all disease-free subjects diagnosed by the gold standard, the proportion of subjects who have negative detection results of the autoantibody or the autoantibody combination accounts for all disease-free subjects.
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. The sample collection has been informed by the subject or patient and approved by the regulatory agency.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.
Example 1
Screening for tumor autoantibody markers was performed using a discovery cohort, including 47 healthy subjects and 41 esophageal cancer patients. Serum samples of esophageal cancer patients are from tumor hospitals in Shanghai city, and serum samples of health examination population are from tumor hospitals in Shanghai city and examination centers.
All esophageal cancer patient sera were collected at the time the patient was initially diagnosed as esophageal cancer and had not received any chemoradiotherapy and surgery and were stored at-80 ℃ freezer.
The patient information used for the discovery cohort is shown in table 1.
Table 1: discovery cohort patient profile
The method comprises the steps of selecting 17 esophageal cancer related antigens, performing expression and purification, coating the antigens on the surface of a 96-well plate, sealing the plate, reacting with esophageal cancer patient serum or normal control human serum diluted by 1:110 times, reacting with an anti-human IgG antibody marked with HRP (horse radish peroxidase), performing color reaction, and detecting with an enzyme labeling instrument OD450nm wavelength. Table 2 shows the sensitivity and specificity of the autoantibodies corresponding to each antigen.
Table 2: discovery of sensitivity and specificity of cohort individual tumor autoantibody markers
| Antigens | Serial number | Sensitivity of the composition | Specificity of |
| 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％ |
The autoantibodies corresponding to the 17 antigens were subjected to three-layer sorting as follows:
1. in previous researches, the inventor finds that the control population has larger bias due to the fact that the samples of the physical examination population from different areas or hospitals are from physical examination, so that the control population using a plurality of different places or hospitals can more accurately represent the actual scene of clinical application, thereby making the detection more clinically meaningful. Therefore, in this study, 90 new healthy control populations were used to perform serum tests for the above tumor antigen autoantibodies, eliminating antigens with a specificity of 94% or less in the new control sera, such as BRCA1(1-110), BORIS.
2. Some antigens have higher sensitivity, but have no single positive contribution rate due to overlapping positive detection with other antigens, so a part of antigens, such as HSP105, Annexin1, SOX2, PRDX1, TROP2 and the like, are eliminated.
Taken together, a preferred set of autoantibody combinations was selected, including autoantibodies against the following 8 tumor associated antigens: trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4 DIP. These 8 autoantibodies were serologically detected in the discovery cohort using the corresponding 8 tumor antigens, and the sensitivity and specificity of the combination of these 8 autoantibodies were found to be 70.7% and 87.0%, respectively (table 3); the detection sensitivity in T0, T1, T2 and T3 stages was 50%, 88%, 63% and 50%, respectively, as analyzed by esophageal cancer stage (Table 4).
Table 3: detection results for finding cohort tumor autoantibody combinations
| Number of people | Number of positives | Sensitivity of the composition | Specificity of |
| 41 (patient) | 29 | 70.7％ | |
| 47 (health) | 6 | 87.0％ |
Table 4: detection sensitivity of different stages of combination of tumor autoantibodies in cohort is discovered
| Staging | Number of patients | Number of positives | Sensitivity (%) |
| T0 | 4 | 2 | 50 |
| T1 | 8 | 7 | 88 |
| T2 | 8 | 5 | 63 |
| T3 | 6 | 3 | 50 |
| Is unknown | 15 | 12 | 80 |
Example 2
The validation cohort included 129 esophageal cancer patients (118 , 6 poorly differentiated, 2 adenocarcinomas, 3 neuroendocrine carcinomas) newly collected from tumor hospitals in Shanghai city, and 88 physical population collected from tumor hospitals and physical centers in Shanghai city, for validation of antibody markers. The patient information is shown in table 5.
Table 5: verification queue patient profile
The level distribution of each tumor autoantibody in the tumor group and the control group against the antigens Trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4DIP are shown as scatter plots (see fig. 1). Antibody level distribution of tumor autoantibodies in tumor and control groups statistical analysis using Mann Whitney test found that autoantibodies against tumor antigens CTAG2, Trim21 and RALA had significantly different level distributions in tumor and control groups (p < 0.05); whereas the level distribution of autoantibodies against the tumor antigens CIP2A, CCDC110, CAGE, MAGEA4 and PDE4DIP were not significantly different between the tumor group and the control group. However, if the levels of autoantibody markers analyzed to be greater than the cutoff values in the tumor group and the control group were distributed in both groups of the population, they were very significantly different. Because of this, all these positive markers above the cutoff value are of great importance for the 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 in the validation cohort are shown in table 6. The specificity of each single marker is 96.6% or more, and the sensitivity is 7.0-17.1%.
Table 6: verification of detection sensitivity and specificity of a cohort of individual autoantibodies
| 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, and the results of the detection of the corresponding tumor autoantibody combinations by these different antigen combinations are shown in table 7. It can be seen that the sensitivity of detection increases and the specificity decreases with increasing number of antigens. The sensitivity and specificity of the three autoantibody combinations (anti-CCDC 110, MAGEA4 and PDE4DIP) detection were 41.5% and 93.6%, respectively, and have reached a considerable level. Adding an autoantibody, such as anti-CTAG 2, to the combination of the three autoantibodies increased the detection sensitivity to 53.6%, and the specificity remained unchanged. Furthermore, with the continued addition of different autoantibody markers, the resulting autoantibody combinations are more sensitive, while the specificity remains around 90% or even higher. For example, increasing 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 combination of autoantibody markers comprising the combination of autoantibodies against the 8 tumour antigens Trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4DIP were 70.7% and 87.2% respectively in the validation cohort.
Table 7: verification of sensitivity and specificity of detection of autoantibody markers for different tumor antigen combinations in cohort
Example 3
Autoantibody marker combinations against 8 tumor antigens Trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4DIP were selected for sensitivity analysis of different stages and typing of esophageal cancer.
Referring to example 2, the validation cohort included 129 esophageal cancer patients (118 , 6 poorly differentiated, 2 adenocarcinomas, 3 neuroendocrine cancers). The validation cohort 129 patients with esophageal cancer included 3T 0 and 25T 1 with sensitivity of 0% and 32%, respectively; the sensitivity of 18 cases T2 and 58 cases T3 is 72% and 60%, respectively; the remaining 25 cases had no information on the stage and the sensitivity was 60%. The results are shown in Table 8.
Table 8: validation of sensitivity of different stages of cohort autoantibody combinations
The validation cohort was mostly carcinomas, 118 cases. Although the incidence of squamous cell carcinoma is reduced and the incidence of adenocarcinoma is increased in the developed western countries, most of the current Chinese esophageal cancer patients still have carcinoma. From the results of the detection, it was found that the combination of 8 autoantibodies preferable in the present invention had a sensitivity of 53% for detection of squamous cell carcinoma. In addition, the positive detection rates for the combination of autoantibodies were 100% for the validation cohort that included 2 adenocarcinomas, 3 neuroendocrine carcinomas. In 6 cases of poorly differentiated cancers, the detection sensitivity was 67%. The results are shown in Table 9.
Table 9: validation of sensitivity of different subtypes of cohort autoantibody combinations
| Subtype of tumor | Number of patients | Sensitivity (%) |
| Adenocarcinoma | 2 | 100 |
| Cancer of neuroendocrine | 3 | 100 |
| Poorly differentiated cancer | 6 | 67 |
| carcinoma | 118 | 53 |
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
Claims (11)
1. A biomarker for esophageal cancer, the biomarker being an autoantibody combination comprising at least three of the autoantibodies against the following tumor antigens, respectively: trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4 DIP.
2. The biomarker of claim 1, wherein the combination of autoantibodies comprises autoantibodies against the following tumor antigens: CCDC110, MAGEA4, and PDE4 DIP;
preferably, the combination of autoantibodies 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;
more preferably, the combination of autoantibodies comprises autoantibodies against the following tumor antigens: CTAG 2;
further preferably, the combination of autoantibodies further comprises autoantibodies against the following tumor antigens: trim21 and RALA; and/or, CIP2A and CAGE.
3. The biomarker of claim 1 or 2, wherein the combination of autoantibodies comprises autoantibodies against the following tumor antigens respectively:
(1) CCDC110, MAGEA4, and PDE4 DIP;
(2) CCDC110, CTAG2, MAGEA4, and PDE4 DIP;
(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.
4. The biomarker of any one of claims 1 to 3, wherein the autoantibody is in whole blood, serum, plasma, tissue or cell, interstitial fluid, cerebrospinal fluid or urine of the subject; wherein preferably, the tissue or cell is esophageal tissue or cell, esophageal cancer tissue or cell, or a tissue or cell adjacent to esophageal cancer;
preferably, the subject is a mammal, preferably a primate mammal, more preferably a human;
preferably, the autoantibody is IgA, IgM or IgG;
preferably, the esophageal cancer comprises esophageal squamous carcinoma, esophageal low-differentiation cancer, esophageal adenocarcinoma, and esophageal neuroendocrine cancer; alternatively, the esophageal cancer comprises stage 0, stage I, stage II, and stage III.
5. A reagent for detecting a biomarker of any of claims 1 to 4.
6. The reagent of claim 5, wherein the reagent is a reagent for enzyme-linked immunosorbent assay (ELISA), protein/peptide chip detection, immunoblotting, microbead immunoassay, or microfluidic immunoassay;
preferably, the reagents are used to detect the biomarkers by antigen-antibody reaction, for example by ELISA or fluorescent or chemiluminescent immunoassay.
7. The agent according to claim 5 or 6, characterized in that it is an antigenic protein combination comprising at least three of the following tumor antigens: trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4 DIP;
preferably, the antigenic protein combination comprises the following tumor antigens: CCDC110, MAGEA4, and PDE4 DIP;
more preferably, the antigenic 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;
more preferably, the antigenic protein combination comprises the following tumor antigens: CTAG 2;
further preferably, the combination of autoantibodies further comprises autoantibodies against the following tumor antigens: trim21 and RALA; and/or, CIP2A and CAGE;
further preferably, the antigenic protein combination comprises the following tumor antigens:
(1) CCDC110, MAGEA4, and PDE4 DIP;
(2) CCDC110, CTAG2, MAGEA4, and PDE4 DIP;
(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.
8. Use of a biomarker according to any of claims 1 to 4 or a reagent according to any of claims 5 to 7 in the manufacture of a product for use in the prediction of risk of developing esophageal cancer, screening, prognostic assessment, monitoring of therapeutic effect or detection of recurrence.
9. The use according to claim 8, wherein the esophageal cancer comprises esophageal squamous carcinoma, esophageal low-differentiation cancer, esophageal adenocarcinoma, and esophageal neuroendocrine cancer; alternatively, the esophageal cancer comprises 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 immunodetection or microfluidic immunodetection; preferably, the kit is used for detecting the biomarkers by antigen-antibody reaction, for example, an ELISA kit or a fluorescent or chemiluminescent immunoassay kit.
10. A kit comprising the reagent of any one of claims 5 to 7.
11. The kit according to claim 10, wherein the kit is a kit for enzyme-linked immunosorbent assay (ELISA), protein/peptide chip detection, immunoblotting, microbead immunoassay, or microfluidic immunoassay; preferably, the kit is used for detecting the biomarkers by antigen-antibody reaction, for example, an ELISA kit or a fluorescent or chemiluminescent immunoassay kit.
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| CN113671180A (en) * | 2021-09-16 | 2021-11-19 | 郑州大学 | Application of PAIP1 autoantibody in auxiliary diagnosis of esophageal squamous cell carcinoma |
| CN114167059A (en) * | 2021-11-03 | 2022-03-11 | 郑州大学 | Biomarker for diagnosing esophageal squamous carcinoma and detection kit |
| WO2022083673A1 (en) * | 2020-10-21 | 2022-04-28 | 杭州凯保罗生物科技有限公司 | Biomarker for esophageal cancer, and use thereof |
| CN115372616A (en) * | 2022-07-15 | 2022-11-22 | 杭州凯保罗生物科技有限公司 | Gastric cancer related biomarker and application thereof |
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