WO2022083673A1 - 食管癌的生物标志物及其应用 - Google Patents

食管癌的生物标志物及其应用 Download PDF

Info

Publication number
WO2022083673A1
WO2022083673A1 PCT/CN2021/125167 CN2021125167W WO2022083673A1 WO 2022083673 A1 WO2022083673 A1 WO 2022083673A1 CN 2021125167 W CN2021125167 W CN 2021125167W WO 2022083673 A1 WO2022083673 A1 WO 2022083673A1
Authority
WO
WIPO (PCT)
Prior art keywords
esophageal cancer
esophageal
autoantibody
ccdc110
ctag2
Prior art date
Application number
PCT/CN2021/125167
Other languages
English (en)
French (fr)
Inventor
孙苏彭
周兴宇
隗啸南
杨盼盼
周静
孙立平
Original Assignee
杭州凯保罗生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 杭州凯保罗生物科技有限公司 filed Critical 杭州凯保罗生物科技有限公司
Publication of WO2022083673A1 publication Critical patent/WO2022083673A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids

Definitions

  • the present invention relates to the field of biotechnology and medical diagnosis, in particular, the present invention relates to an autoantibody biomarker of esophageal cancer, an antigen combination for detecting the same, and their corresponding application in the detection of esophageal cancer.
  • Esophageal cancer is a common malignant tumor worldwide. China is a country with a high incidence of esophageal cancer, with 230,000 new cases every year, accounting for about half of the world’s Although the incidence of esophageal cancer is significantly lower than that in China, there are as many as 17,000 new cases every year. Risk factors for esophageal cancer include smoking, drinking, eating too little fruits and vegetables, esophageal reflux, obesity and smoking, etc. Age, gender (male) and family history also account for certain risk factors.
  • Esophageal cancer has no obvious feeling in the early stage and will not attract people's attention; once clinical signs such as dysphagia, retrosternal pain or discomfort appear, about 90% of patients are already in the middle and late stages, and the treatment effect is poor. Therefore, it is of great clinical significance to effectively screen the early lesions of esophageal cancer.
  • endoscopy-biopsy is mainly used for early detection of esophageal cancer in high-risk groups.
  • different operator habits or incorrect biopsy sampling can cause detection errors.
  • the early manifestation of esophageal endothelial cell hyperplasia is a pudding-like distribution, which is unevenly distributed in the biopsy tissue, which increases the difficulty of early diagnosis.
  • monitoring the proliferation of esophageal epithelial cells is not necessarily effective in early diagnosis of esophageal cancer.
  • tumor antigen markers such as tumor antigen markers, circulating DNA, methylated fragments or miRNA to detect esophageal cancer.
  • markers are usually derived directly from tumor cells and enter the circulatory system from the tumor as a result of tumor cell death or active signaling. Due to the small number of cancer cells in early stage tumors, only a few of them die and actively release, coupled with the short half-life of these markers in the circulatory system, this signal from the tumor is very weak or even non-existent and cannot be used as a good early stage tumor marker. At the same time, because the detection steps of such markers are too many or the standardization is not enough, the clinical application has the defects of difficulty, long cycle and high cost.
  • Autoantibodies are antibodies directed against one's own tissues, organs, cells and cellular components.
  • the exposure of tumor-associated antigens can be recognized by the human immune system, resulting in tumor-associated autoantibodies, and even maintain high levels of autoantibodies in peripheral blood, which can be sensitively detected by conventional technical means in the art.
  • autoantibodies produced by tumor antigens are good indicators for early diagnosis of tumors. Using tumor-induced autoantibodies to reflect the tumorigenesis mechanism and disease progression of patients is becoming a new target for early diagnosis and prognosis judgment of tumors. important direction.
  • the combination of several autoantibodies for the clinical diagnosis of esophageal cancer can significantly improve the accuracy of early diagnosis of esophageal cancer, and the earlier the esophageal cancer is detected, the greater the significance of clinical treatment. Therefore, in order to solve the above technical problems, the present invention finally identifies a group of autoantibodies that can be used for screening esophageal cancer, especially early esophageal cancer, by detecting autoantibodies against different tumor antigen targets in the blood of esophageal cancer patients.
  • the autoantibody combination has sufficiently high sensitivity especially in early stage tumors, especially in the experimental Chinese population, and also has sufficiently high detection specificity.
  • an object of the present invention is to provide a biomarker for esophageal cancer, which is a combination of autoantibodies.
  • another object of the present invention is to provide a reagent for detecting the autoantibody combination, such as an antigen protein combination; Use in products such as disease risk prediction, screening, prognosis assessment, treatment effect monitoring or recurrence monitoring.
  • Another object of the present invention is to provide a kit and a corresponding method for risk prediction, screening, prognosis evaluation, treatment effect monitoring or recurrence monitoring of esophageal cancer.
  • the present invention provides a biomarker for esophageal cancer
  • the biomarker is a combination of autoantibodies including at least three autoantibodies against the following tumor antigens: Trim21, CIP2A, CTAG2, CCDC110, CAGE , MAGEA4, RALA, and PDE4DIP.
  • the autoantibody combination comprises autoantibodies against the following tumor antigens, respectively: CCDC110, MAGEA4 and PDE4DIP.
  • 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 .
  • the autoantibody combination preferably includes autoantibodies against the following tumor antigens: CTAG2.
  • the autoantibody combination further includes autoantibodies against the following tumor antigens: Trim21 and RALA; and/or, CIP2A and CAGE.
  • the autoantibody combination comprises autoantibodies against the following tumor antigens, respectively:
  • the autoantibody is an autoantibody 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 Cells, esophageal cancer tissue or cells, or paracancerous tissue or cells of esophageal cancer.
  • the subject is a mammal, preferably a primate, more preferably a human.
  • the subject is a Chinese population.
  • the autoantibody is IgA (eg IgAl, IgA2), IgM or IgG (eg IgGl, IgG2, IgG3, IgG4).
  • the esophageal cancer includes esophageal squamous cell carcinoma, poorly differentiated esophageal carcinoma, esophageal adenocarcinoma and esophageal neuroendocrine carcinoma; or, the esophageal cancer includes stage 0, stage I, stage II, and stage III. According to a specific embodiment of the invention, esophageal cancer is staged according to the pTNM system.
  • the combination of biomarkers ie autoantibodies
  • a sample eg plasma or serum
  • autoantibodies can be used interchangeably with "positive” or “negative”; it is a routine technique in the art to judge this.
  • Detection can be performed, for example, by antigen-antibody-specific reactions between tumor-associated antigens that result in the appearance of any autoantibodies in the combination.
  • the present invention also provides a reagent for detecting the biomarkers of the present invention.
  • the reagents may be reagents for enzyme-linked immunosorbent assay (ELISA), protein/peptide chip detection, immunoblotting, microbead immunoassay, or microfluidic immunoassay, and the like.
  • ELISA enzyme-linked immunosorbent assay
  • the reagents are used to detect the biomarkers of the invention by antigen-antibody reaction, eg by ELISA or fluorescence or chemiluminescence immunodetection.
  • the agent may be an antigenic protein combination comprising at least three of the following tumor antigens: Trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA, and PDE4DIP.
  • the antigen-protein combination includes the following tumor antigens: CCDC110, MAGEA4 and PDE4DIP.
  • 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.
  • the antigen-protein combination preferably includes the following tumor antigen: CTAG2.
  • the autoantibody combination also includes autoantibodies against the following tumor antigens: Trim21 and RALA; and/or, CIP2A and CAGE;
  • the antigen-protein combination includes the following tumor antigens:
  • the present invention provides the use of the biomarker or reagent in the preparation of a product for risk prediction, screening, prognosis evaluation, treatment effect monitoring or recurrence monitoring of esophageal cancer.
  • the esophageal cancer includes esophageal squamous cell carcinoma, poorly differentiated esophageal carcinoma, esophageal adenocarcinoma, and esophageal neuroendocrine carcinoma; or, the esophageal cancer includes stage 0, stage I, stage II, and stage III esophageal cancer.
  • the product is a kit; more preferably, the kit is used for enzyme-linked immunosorbent assay (ELISA), protein/peptide chip detection, western blotting, bead immunoassay or microfluidic immunoassay the kit.
  • the kit is used to detect the biomarker through antigen-antibody reaction, such as an ELISA kit or a fluorescence or chemiluminescence immunodetection kit.
  • the present invention provides a kit comprising the reagent of the present invention.
  • the kit may be a kit for enzyme-linked immunosorbent assay (ELISA), protein/peptide chip detection, immunoblotting, microbead immunoassay, or microfluidic immunoassay, and the like.
  • ELISA enzyme-linked immunosorbent assay
  • the kit is used to detect the biomarkers of the present invention by antigen-antibody reaction, such as an ELISA kit or a fluorescence or chemiluminescence immunodetection kit.
  • the kit is an enzyme-linked immunosorbent assay (ELISA) detection kit. That is, using the kit, whether the autoantibody biomarker is positive in the sample of the subject is detected by enzyme-linked immunosorbent assay.
  • the kit may also include other components required for ELISA detection of autoantibody biomarkers, which are well known in the art.
  • the antigenic protein in the kit can be linked with a tag peptide, such as His tag, streptavidin tag, Myc tag; for another example, the kit can include a solid phase carrier, such as with an immobilized antigen. Protein microwell carriers, such as microtiter plates; or microbeads or magnetic bead solid-phase carriers.
  • It can also include adsorbed proteins for immobilizing antigen proteins on solid supports, dilutions of blood such as serum, washing solutions, secondary antibodies with enzyme-labeled or fluorescent or chemiluminescent substances, chromogenic solutions, Stop solution, etc.
  • 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 to form an antigen-antibody complex.
  • the present invention provides a method for disease risk prediction, screening, prognosis evaluation, treatment effect monitoring or recurrence monitoring of esophageal cancer, comprising the following steps:
  • the quantification includes detecting each autoantibody in the autoantibody combination using the reagent provided by the present invention (ie, the antigen-protein combination) or a kit comprising the reagent.
  • the subject is a mammal, preferably a primate mammal, more preferably a human.
  • the esophageal cancer includes esophageal squamous cell carcinoma, esophageal poorly differentiated carcinoma, esophageal adenocarcinoma, and esophageal neuroendocrine carcinoma; or, the esophageal cancer includes stage 0, stage I, stage II, and stage III esophageal cancer.
  • the sample is 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 cells or adjacent tissue or cells of esophageal cancer.
  • the reference threshold may be a reference level from a healthy person or a healthy population; for example, it may be defined as the mean plus 2 standard deviations or 3 standard deviation.
  • the present invention provides a novel biomarker for esophageal cancer, which is a completely new group of tumor autoantibodies.
  • Each of these autoantibodies has a separate positive contribution rate for the detection of esophageal cancer, and has a fairly high detection specificity and sensitivity. When used in combination, it has a fairly high detection sensitivity and detection specificity, even reaching 50% sensitivity in the case of stage 0 esophageal cancer.
  • Figure 1 is a scatter plot of the level distribution of each tumor autoantibody in the tumor group and the control group.
  • the term "antigen” or the term “antigenic protein” are used interchangeably.
  • the following experimental procedures or definitions are involved in the present invention. It should be noted that the present invention can also be implemented using other conventional techniques in the art, and is not limited to the following experimental operations.
  • the cDNA fragment of tumor antigen was cloned into PET28(a) expression vector containing 6XHis tag.
  • a streptavidin protein or an analog (a biotin-binding tag protein) is introduced.
  • the obtained recombinant expression vector was transformed into E. coli for expression.
  • the proteins expressed in the supernatant were purified by Ni-NTA affinity column and ion column.
  • the protein is denatured with 6M guanidine hydrochloride, renatured and folded according to standard methods in vitro, and then purified by Ni-NTA affinity column through 6XHis tag to obtain the antigenic protein.
  • Serum or plasma from patients with esophageal cancer were collected when the patients were initially diagnosed with esophageal cancer and had not received any radiotherapy, chemotherapy or surgery. Plasma or serum were prepared according to standard clinical procedures and stored in a -80°C freezer for long-term storage.
  • the concentration of autoantibodies in the samples was quantified by enzyme-linked immunosorbent assay (ELISA).
  • Microwells are pre-coated with biotinylated bovine serum albumin (BSA).
  • BSA biotinylated bovine serum albumin
  • Serum or plasma samples were diluted 1:110 with phosphate buffer and added to microwells for reaction (50 mL/well). After washing unbound serum or plasma fractions with wash solution, horseradish peroxidase (HRP)-conjugated anti-human IgG was added to each well for the reaction. Then the reaction substrate TMB (3,3',5,5'-tetramethylbenzidine) was added for color development. A stop solution (1N HCl) was added, and the absorbance at 450nm was single-spectrum for reading (OD) by a microplate reader. Serum autoantibody concentrations were quantified using a standard curve.
  • the cutoff value for autoantibodies was defined as the mean plus 2 standard deviations (SD) or the mean plus 3 standard deviations (SD) of the detected absorbance values in a control normal population confirmed by physical examination to be free of cancer. Which of the above two cases is selected to determine the cutoff value of each autoantibody is based on the following principles: 1. When using two different values (mean plus 2 standard deviations and mean plus 3 standard deviations) as a reference In the case of the threshold value, the detection specificity of each autoantibody to the control normal population is 95% or above; 2. In the case of using the two different values as the reference threshold value, each autoantibody pair is obtained. The specificity of the control normal population and the sensitivity to the esophageal cancer patient population were described, the sum of the two was calculated, and the value when the sum was larger was selected as the determined cutoff value of the autoantibody.
  • SD standard deviations
  • SD standard deviations
  • a positive reaction was defined as a quantification of the level of autoantibodies in the sample, which was compared to the cutoff value, with a ⁇ cutoff value of positive; correspondingly, a negative reaction was defined as a ⁇ cutoff value of negative.
  • the results of multiple autoantibodies are combined to judge the prediction effect when analyzing the results.
  • the rule is: if multiple autoantibodies are detected in the sample, as long as one or more of the autoantibodies are positive, the result of the combination of antibodies is judged to be positive; and if all the autoantibodies are negative, the result of the combination of antibodies is judged to be negative.
  • Sensitivity Among all cases of esophageal cancer diagnosed by the gold standard, the proportion of cases with positive autoantibody or autoantibody combination test results in all cases.
  • Serum from all esophageal cancer patients was collected when the patients were initially diagnosed with esophageal cancer and had not received any radiotherapy, chemotherapy and surgery, and was stored in a -80 degree refrigerator.
  • the patient information used for the discovery cohort is shown in Table 1.
  • Table 1 Discovery cohort patient characteristics table
  • 17 esophageal cancer-related antigens were selected to be expressed and purified, and then coated on the surface of 96-well plate. After blocking, the serum of patients with esophageal cancer or the serum of normal control population was diluted 1:110 times, and then reacted with the labeled HRP horseradish catalase. The anti-human IgG antibody was reacted, and then the color reaction was carried out and detected with a microplate reader OD450nm wavelength. Table 2 shows the sensitivity and specificity of autoantibodies corresponding to each antigen.
  • the autoantibodies corresponding to the above 17 antigens were sorted in the following three layers:
  • the autoantibodies of some antigens have high sensitivity, due to the overlapping positive detection with the autoantibodies of other antigens, there is no independent positive contribution rate, so the autoantibodies of some antigens, such as HSP105, Annexin1, SOX2, PRDX1 and TROP2, etc.
  • autoantibodies against the following 8 tumor-associated antigens Trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4DIP.
  • Serological testing of these 8 autoantibodies in the discovery cohort using the corresponding 8 tumor antigens found that the sensitivity and specificity of the combination of these 8 autoantibodies were 70.7% and 87.0%, respectively (Table 3); Cancer staging was analyzed, and the detection sensitivities at T0, T1, T2, and T3 stages were 50%, 88%, 63%, and 50%, respectively (Table 4).
  • the validation cohort included 129 esophageal cancer patients (118 squamous cell carcinomas, 6 poorly differentiated carcinomas, 2 adenocarcinomas, and 3 neuroendocrine carcinomas) newly collected from Shanghai Cancer Hospital, and from Shanghai Cancer Hospital and Physical Examination Center. The verification of antibody markers was carried out in 88 cases of physical examination population. Patient information is shown in Table 5.
  • Table 5 Validation cohort patient characteristics table
  • the level distribution of each tumor autoantibody in the tumor group and the control group was represented by a scatter plot (see Figure 1).
  • the antibody level distribution of tumor autoantibodies in the tumor group and the control group was statistically analyzed using the Mann Whitney test, and it was found that the level distribution of autoantibodies against the tumor antigens CTAG2, Trim21 and RALA was significantly different between the tumor group and the control group (p ⁇ 0.05)
  • the levels of autoantibodies against tumor antigens CIP2A, CCDC110, CAGE, MAGEA4 and PDE4DIP were not significantly different between tumor and control groups.
  • Table 6 shows the detection sensitivity and specificity of 8 tumor autoantibody markers in the sera of 129 esophageal cancer patients and 88 healthy controls in the validation cohort.
  • the specificity of each marker was 96.6% or above, and the sensitivity was between 7.0% and 17.1%.
  • the resulting autoantibody combination is more sensitive, while the specificity remains around 90% or even higher.
  • the increased sensitivity and specificity of Trim21 and RALA detection on this basis were 65.8% and 89.3%, respectively; while including autoantibodies against 8 tumor antigens Trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4DIP
  • the sensitivity and specificity of the marker combination in the validation cohort were 70.7% and 87.2%, respectively.
  • a combination of autoantibody markers against 8 tumor antigens Trim21, CIP2A, CTAG2, CCDC110, CAGE, MAGEA4, RALA and PDE4DIP were selected for sensitivity analysis of different stages and types of esophageal cancer.
  • the validation cohort included 129 patients with esophageal cancer (118 squamous cell carcinomas, 6 poorly differentiated carcinomas, 2 adenocarcinomas, 3 neuroendocrine carcinomas).
  • the validation cohort of 129 patients with esophageal cancer included 3 patients with T0 stage and 25 patients with T1 stage, the detection sensitivity was 0% and 32%, respectively; 18 patients with T2 stage and 58 patients with T3 stage, the sensitivity was 72% and 60%, respectively; The remaining 25 cases had no staging information, with a sensitivity of 60%.
  • Table 8 The results are shown in Table 8.
  • the validation cohort was mostly squamous cell carcinoma with 118 cases. Although the incidence of squamous cell carcinoma in esophageal cancer in western developed countries has decreased and the incidence of adenocarcinoma has increased, the majority of esophageal cancer patients in China are still squamous cell carcinoma. It can be seen from the detection results that the combination of the eight autoantibodies preferred in the present invention has a detection sensitivity of 53% for squamous cell carcinoma. In addition, the validation cohort included 2 adenocarcinomas and 3 neuroendocrine carcinomas, both with 100% positive detection rates for the autoantibody combination. There were also 6 cases of poorly differentiated carcinoma, which had a detection sensitivity of 67%. The results are shown in Table 9.
  • adenocarcinoma 2 100 neuroendocrine carcinoma 3 100 poorly differentiated carcinoma 6 67 squamous cell carcinoma 118 53

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种食管癌的生物标志物，生物标志物为自身抗体组合，包括分别抗以下肿瘤抗原的自身抗体中的至少三个：Trim21、CIP2A、CTAG2、CCDC110、CAGE、MAGEA4、RALA和PDE4DIP；通过检测生物标志物能够对食管癌进行早期筛查。一种用于检测生物标志物的抗原蛋白组合以及包含抗原蛋白组合的试剂盒，以及相应的检测或诊断方法。

Description

食管癌的生物标志物及其应用
相关申请的交叉引用
本专利申请要求于2020年10月21日提交的申请号为CN2020111304307的中国发明专利申请的优先权权益，在此将其全部内容引入作为参考。
技术领域
本发明涉及生物技术和医学诊断领域，具体而言，本发明涉及一种食管癌的自身抗体生物标志物、检测其的抗原组合以及相应地它们在食管癌检测中的应用。
背景技术
食管癌是一种全球常见的恶性肿瘤。中国是食管癌高发国家，每年新增病例23万，约占全球一半，发病率位居恶性肿瘤的第5位，死亡率位居第4位，且发病率有低龄化趋势；而欧美发达国家食管癌的发病率虽然显著低于中国，但每年新增病例也有1.7万之多。食管癌的高危因素包括吸烟、饮酒、过少进食水果蔬菜、食管反流、肥胖和吸烟等等；年龄、性别(男性)和家族史也占一定风险因素。
食道癌在发病初期并没有什么明显的感觉，不会引起人们的注意；一旦出现吞咽困难、胸骨后疼痛或不适等临床征兆时，约有90％的患者已经是中晚期，治疗效果差。因此，有效筛查食管癌的早期病变具有很大的临床意义。
当前，主要采用内镜检查-活检对高危人群进行食管癌的早期检查。但是，操作者的习惯不同或者活检取样失误，会造成检测失误。并且，食管内皮细胞组织增生在早期的表现是布丁式分布，在活检组织中分布不均匀，增加早期诊断的难度。同时，监视食管上皮细胞的增生也并不一定能有效地早期诊断出食管癌。
目前还已提出了利用血液标志物如肿瘤抗原标志物、循环DNA、甲基化片段或者miRNA等来进行食管癌的检测。这类标志物通常直接来自于肿瘤细胞，由于肿瘤细胞死亡或主动释放信号而从肿瘤进入到循环系统中。由于早期肿瘤的癌症细胞数量少，只有其中少数细胞死亡和主动释放，再加上这些标志物在循环系统的半衰期短，因此这种来自肿瘤的信号非常弱甚至没有，无法作为很好的早期肿瘤的标志物。同时，由于这类标志物的检测步骤 多或标准化不够，使得在临床上的应用具有难度大、周期长、费用高等缺陷。
自身抗体是指针对自身组织、器官、细胞及细胞成分的抗体。在癌症发生早期，肿瘤相关抗原的暴露即可被人体免疫系统识别，产生肿瘤相关自身抗体，甚至在外周血中也可维持高水平的自身抗体，可被本领域常规技术手段灵敏地检测到。本领域公认，肿瘤抗原产生的自身抗体是肿瘤早期诊断的较好指标，利用肿瘤诱导产生的自身抗体来反应患者肿瘤发生机制、疾病进展过程，正成为寻找用于肿瘤早期诊断及预后判断新靶标的重要方向。
已有研究提出将自身抗体检测用于食管癌筛查中。但是，由于肿瘤的异质性和不同个体间免疫系统反应的不同，单个肿瘤自身抗体在肿瘤病人中的敏感性不够高，通常仅为5％～20％。因此，联合使用多个不同的肿瘤自身抗体组合才能增加检测敏感性。
发明内容
将几种自身抗体联合用于食管癌的临床诊断，可以显著提高诊断早期食管癌的准确度，而越早发现食管癌，临床治疗意义越大。因此，为了解决上述技术问题，本发明通过检测食管癌患者血液中针对不同肿瘤抗原靶点的自身抗体，最终鉴定了一组可用于筛查食管癌、尤其是早期食管癌的自身抗体。该自身抗体组合作为生物标志物，在特别是早期肿瘤中有足够高的敏感性，尤其是在实验的中国人群中；同时还具有足够高的检测特异性。
因此，本发明的一个目的是提供一种食管癌的生物标志物，其为自身抗体组合。
基于该自身抗体组合作为生物标志物，本发明的另一个目的是提供用于检测该自身抗体组合的试剂，例如抗原蛋白组合；以及提供该自身抗体组合或检测试剂在制备用于食管癌的患病风险预测、筛查、预后评估、治疗效果监测或复发监测等产品中的用途。
本发明的再一个目的是提供试剂盒以及相应地用于食管癌的患病风险预测、筛查、预后评估、治疗效果监测或复发监测等的方法。
本发明的技术方案如下。
一方面，本发明提供一种食管癌的生物标志物，所述生物标志物为自身抗体组合，其包括分别抗以下肿瘤抗原的自身抗体中的至少三个：Trim21、CIP2A、CTAG2、CCDC110、CAGE、MAGEA4、RALA和PDE4DIP。
优选地，所述自身抗体组合包括分别抗以下肿瘤抗原的自身抗体： CCDC110、MAGEA4和PDE4DIP。
更优选地，所述自身抗体组合还包括分别抗以下肿瘤抗原的自身抗体中的至少一个、至少两个、至少三个、至少四个或甚至至少五个：CTAG2、Trim21、CIP2A、CAGE和RALA。其中，所述自身抗体组合优选地包括抗以下肿瘤抗原的自身抗体：CTAG2。
进一步优选地，所述自身抗体组合还包括抗以下肿瘤抗原的自身抗体：Trim21和RALA；和/或，CIP2A和CAGE。
根据本发明的具体实施方式，所述自身抗体组合包括分别抗以下肿瘤抗原的自身抗体：
(1)CCDC110、MAGEA4和PDE4DIP；
(2)CCDC110、CTAG2、MAGEA4和PDE4DIP；
(3)CCDC110、CTAG2、MAGEA4、PDE4DIP、Trim21和RALA；
(4)CCDC110、CTAG2、MAGEA4、PDE4DIP、CIP2A和CAGE；
(5)CCDC110、CTAG2、MAGEA4、PDE4DIP、Trim21、RALA、CIP2A和CAGE。
根据本发明，所述自身抗体为受试者的全血、血清、血浆、组织或细胞、组织间隙液、脑脊液或尿液中的自身抗体；其中优选地，所述组织或细胞为食管组织或细胞、食管癌组织或细胞或者食管癌的癌旁组织或细胞。
优选地，所述受试者为哺乳动物，优选为灵长类哺乳动物，更优选为人。例如，所述受试者为中国人群。
优选地，所述自身抗体为IgA(例如IgA1、IgA2)、IgM或IgG(例如IgG1、IgG2、IgG3、IgG4)。
优选地，所述食管癌包括食管鳞癌、食管低分化癌、食管腺癌和食管神经内分泌癌；或者，所述食管癌包括0期、I期、II期、III期。根据本发明的具体实施方式，食管癌的分期根据pTNM系统。
根据本发明，该生物标志物即自身抗体组合可以在受试者的样本(例如血浆或血清)中进行检测。在本发明中，自身抗体的“存在”或“不存在”与“阳性”或“阴性”可互换使用；对此进行判断为本领域常规技术。例如，可以通过导致该组合中任何自身抗体出现的肿瘤相关抗原与其之间的抗原抗体特异性反应进行检测。因此相应地，另一方面，本发明还提供一种用于检测本发明的生物标志物的试剂。
取决于具体技术手段，所述试剂可以是用于酶联免疫吸附法(ELISA)、 蛋白/肽段芯片检测、免疫印迹、微珠免疫检测或微流控免疫检测等的试剂。优选地，所述试剂用于通过抗原抗体反应对本发明的生物标志物进行检测，例如通过ELISA或荧光或化学发光免疫检测。
在这一方面，所述试剂可以是抗原蛋白组合，其包括以下肿瘤抗原中的至少三个：Trim21、CIP2A、CTAG2、CCDC110、CAGE、MAGEA4、RALA和PDE4DIP。
优选地，所述抗原蛋白组合包括以下肿瘤抗原：CCDC110、MAGEA4和PDE4DIP。
更优选地，所述抗原蛋白组合还包括以下肿瘤抗原中的至少一个、至少两个、至少三个、至少四个或甚至至少五个：CTAG2、Trim21、CIP2A、CAGE和RALA。其中，所述抗原蛋白组合优选地包括以下肿瘤抗原：CTAG2。进一步优选地，所述自身抗体组合还包括抗以下肿瘤抗原的自身抗体：Trim21和RALA；和/或，CIP2A和CAGE；
根据本发明的具体实施方式，所述抗原蛋白组合包括以下肿瘤抗原：
(1)CCDC110、MAGEA4和PDE4DIP；
(2)CCDC110、CTAG2、MAGEA4和PDE4DIP；
(3)CCDC110、CTAG2、MAGEA4、PDE4DIP、Trim21和RALA；
(4)CCDC110、CTAG2、MAGEA4、PDE4DIP、CIP2A和CAGE；
(5)CCDC110、CTAG2、MAGEA4、PDE4DIP、Trim21、RALA、CIP2A和CAGE。
再一方面，本发明提供所述生物标志物或试剂在制备用于食管癌的患病风险预测、筛查、预后评估、治疗效果监测或复发监测的产品中的用途。
优选地，所述食管癌包括食管鳞癌、食管低分化癌、食管腺癌和食管神经内分泌癌；或者，所述食管癌包括0期、I期、II期、III期食管癌。
优选地，所述产品为试剂盒；更优选地，所述试剂盒是用于酶联免疫吸附法(ELISA)、蛋白/肽段芯片检测、免疫印迹、微珠免疫检测或微流控免疫检测的试剂盒。优选地，所述试剂盒用于通过抗原抗体反应对所述生物标志物进行检测，例如为ELISA试剂盒或荧光或化学发光免疫检测试剂盒。
又一方面，本发明提供一种试剂盒，所述试剂盒包含本发明所述的试剂。
取决于具体技术手段，所述试剂盒可以是用于酶联免疫吸附法(ELISA)、蛋白/肽段芯片检测、免疫印迹、微珠免疫检测或微流控免疫检测等的试剂盒。优选地，所述试剂盒用于通过抗原抗体反应对本发明的生物 标志物进行检测，例如为ELISA试剂盒或荧光或化学发光免疫检测试剂盒。
因此，优选地，试剂盒为酶联免疫吸附法(ELISA)检测试剂盒。即，采用该试剂盒，通过酶联免疫吸附法来检测受试者的样本中自身抗体生物标志物是否是阳性的。相应地，所述试剂盒还可包括用于对自身抗体生物标志物进行ELISA检测的所需其他组分，这些均是本领域公知的。出于检测目的，例如，试剂盒中的抗原蛋白可连接有标签肽，例如His标签、链霉亲和素标签、Myc标签；又如，该试剂盒可以包括固相载体，如具有可固定抗原蛋白的微孔的载体，如酶标板；或者微珠或磁珠固相载体。还可以包括用于将抗原蛋白固定于固相载体上的吸附蛋白、血液如血清的稀释液、洗涤液、带有酶标记的二抗或者荧光或化学发光的物质的二抗、显色液、终止液等。通过间接或直接包被在固相载体表面的抗原蛋白，和血清/血浆/组织液等中的抗体进行反应形成抗原－抗体复合物的原理来检测体液中的相应抗体的浓度。
还一方面，本发明提供一种食管癌的患病风险预测、筛查、预后评估、治疗效果监测或复发监测的方法，其包括以下步骤：
(1)对来自受试者的样本进行本发明提供的自身抗体组合中的每个自身抗体的定量；
(2)将所述自身抗体的量与参考阈值进行比较，在其高于参考阈值时，确定所述受试者具有食管癌患病风险、患有食管癌、预后不良或食管癌治疗效果差。
在步骤(1)中，所述定量包括采用本发明提供的试剂(即抗原蛋白组合)或包含该试剂的试剂盒对所述自身抗体组合中的每个自身抗体进行检测。
根据本发明，所述受试者为哺乳动物，优选为灵长类哺乳动物，更优选为人。并且，优选地，所述食管癌包括食管鳞癌、食管低分化癌、食管腺癌和食管神经内分泌癌；或者，所述食管癌包括0期、I期、II期、III期食管癌。
根据本发明，所述样本为受试者的全血、血清、血浆、组织或细胞、组织间隙液、脑脊液或尿液；其中优选地，所述组织或细胞为食管组织或细胞、食管癌组织或细胞或者食管癌的癌旁组织或细胞。
在步骤(2)中，所述参考阈值可以是来自健康人或健康人群的参照水平；例如，可以被定义为经身体检查确认未患有癌症的人群的平均值加2个标准偏差或3个标准偏差。
与现有技术相比，本发明提供了一种食管癌的新型生物标志物，其为全新的一组肿瘤自身抗体。这些自身抗体中的每一个均对食管癌的检测有单独的阳性贡献率，且具有相当高的检测特异性和敏感性。在组合使用时，具有相当高的检测敏感性和检测特异性，甚至在0期食管癌情况下即可达到50％的敏感性。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为每个肿瘤自身抗体在肿瘤组和对照组的水平分布散点图。
实施发明的最佳方式
在本发明中，术语“抗原”或术语“抗原蛋白”可互换使用。此外，本发明中涉及以下实验操作或定义。应注意，本发明还可采用本领域其他常规技术进行实施，并不仅限于以下实验操作。
(一)重组抗原蛋白的制备
将肿瘤抗原的cDNA片段克隆到含6XHis标记的PET28(a)表达载体上。在抗原的N端或C端，引入链霉亲和素蛋白或类似物(结合生物素的标签蛋白)。获得的重组表达载体转化大肠杆菌进行表达。上清中表达的蛋白通过Ni-NTA亲和柱和离子柱进行纯化。当蛋白表达在包涵体内，用6M盐酸胍对蛋白进行变性处理，并在体外按照标准方法进行复性折叠，然后通过6XHis标签进行Ni-NTA亲和柱纯化，获得抗原蛋白。
(二)血清或血浆的制备和保存
食管癌患者血清或血浆在患者最初诊断为食管癌，尚未接受任何放化疗及手术治疗时收集。血浆或血清按标准临床程序制备，置于-80℃冰箱中长期保存。
(三)ELISA检测
通过酶联免疫吸附测定(ELISA)定量样本中自身抗体的浓度。
纯化的肿瘤抗原通过其标签链霉亲和素或类似物固定到微孔表面。微孔预包被生物素标记的牛血清白蛋白(BSA)。血清或血浆样本用磷酸盐缓冲稀释1:110倍，加入微孔进行反应(50毫升/孔)。用洗液冲洗未结合的血清或血浆成分后，每孔加入辣根过氧化物酶(HRP)偶联的抗-人IgG进行反应。然后加入反应底物TMB(3,3',5,5'-四甲基联苯胺)进行显色。加入终止 溶液(1N HCl)，吸光度为450nm单光谱进行酶标仪进行读值(OD)。用标准曲线定量血清自身抗体浓度。
(四)自身抗体的临界值(cutoff值)
自身抗体的cutoff值被定义为在经身体检查确认未患有癌症的对照正常人群的检测吸光度值的平均值加2个标准偏差(SD)或平均值加3个标准偏差(SD)。从前述两种情况中选择哪种来确定每个自身抗体的cutoff值为根据以下原则：1.在采用两个不同数值(平均值加2个标准偏差和平均值加3个标准偏差)作为参考阈值的情况下，每个自身抗体对所述对照正常人群的检测特异性均为95％或以上；2.在采用所述两个不同数值作为参考阈值的情况下，得到每个自身抗体对所述对照正常人群的特异性和对食管癌患者人群的敏感性，计算二者总和，选择该总和更大情况下的数值作为该自身抗体的确定的cutoff值。
(五)单个自身抗体的阳性、阴性判断
对于每种自身抗体测定，阳性反应定义为对样本中自身抗体的水平进行定量后，将其与cutoff值进行比较，≥cutoff值为阳性；相应地，阴性反应定义为＜cutoff值为阴性。
(六)自身抗体组合的阳性判断
由于单个自身抗体的阳性率低，为了增加自身抗体检出的阳性率，分析结果时联合多个自身抗体的结果来判断预测效果。规则是：在样本中检测多个自身抗体，只要有其中一个或者多个自身抗体显示阳性，则判断抗体组合结果为阳性；而如果所有的自身抗体均为阴性，则判断抗体组合结果为阴性。
(七)统计分析方法
使用GraphPad Prism v.6(Graphpad Prism软件，加利福尼亚州圣地亚哥)和针对Windows的IBM SPSS Statistics 23(IBM，纽约，纽约)，使用Mann–Whitney U检验对两组进行了统计学分析。在分析每个参数之间的关系时，执行了Spearman的相关分析。
(八)敏感性和特异性判断
敏感性：金标准诊断全部食管癌病例中，自身抗体或自身抗体组合检测结果为阳性的病例占全部病例的比例。
特异性：金标准诊断全部无病的受试者中，自身抗体或自身抗体组合检测结果为阴性的受试者占全部无病受试者的比例。
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些 实施例仅用于说明本发明，其不以任何方式限制本发明的范围。样本采集已经受试者或患者知情同意，且获得监管部门的批准。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例1
使用发现队列，包括47例健康体检人群和41例食管癌患者，进行肿瘤自身抗体标志物的筛选。食管癌患者血清标本来自上海市肿瘤医院，健康体检人群血清标本来自上海市肿瘤医院和体检中心。
所有食管癌患者血清都是在患者最初诊断为食管癌尚未接受任何放化疗及手术治疗时收集的，并在-80度冰箱保存。
发现队列所用的患者信息如表1显示。
表1：发现队列患者特征表
Figure PCTCN2021125167-appb-000001
选择17个食管癌相关抗原经过表达纯化后包被到96孔板表面，封闭后和稀释1:110倍的食管癌患者人群血清或正常对照人群血清反应，接着和标记HRP辣根过氧化氢酶的抗人IgG抗体反应，然后进行显色反应，并用酶 标仪OD450nm波长检测。表2显示对应于每个抗原的自身抗体的敏感性和特异性。
表2：发现队列单个肿瘤自身抗体标志物的敏感性和特异性
抗原 序列号 敏感性 特异性
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％
将上述17个抗原对应的自身抗体进行如下三层分选：
1.发明人在以前研究中发现，对照人群由于来自体检，不同区域或医院的体检人群样本会出现较大的偏倚，因此使用多个不同地方或医院的对照人群能更准确地代表临床应用的实际场景，从而使检测更有临床意义。因此，本研究中又使用了90例新的健康对照人群进行上述肿瘤抗原自身抗体的血清检测，去除了在新的对照血清中特异性在94％以下的抗原，比如BRCA1(1-110)、BORIS。
2.有些抗原的自身抗体虽然有较高的敏感性，但由于和其他抗原的自身抗体有重叠的阳性检出，没有单独阳性贡献率，因此剔除了一部分抗原的自身抗体，该抗原如HSP105、Annexin1、SOX2、PRDX1和TROP2等。
经综合考虑，选择一组优选的自身抗体组合，包括分别针对以下8个肿瘤相关抗原的自身抗体：Trim21，CIP2A，CTAG2，CCDC110，CAGE，MAGEA4，RALA和PDE4DIP。使用相应的8个肿瘤抗原在发现队列中对这8个自身抗体进行血清检测，发现这8个自身抗体的组合的敏感性和特异性分别是70.7％和87.0％(表3)；而按照食管癌分期进行分析，在T0、T1、T2、T3期的检测敏感性分别为50％，88％，63％和50％(表4)。
表3：发现队列肿瘤自身抗体组合的检测结果
人群数目 阳性数目 敏感性 特异性
41(患者) 29 70.7％  
47(健康) 6   87.0％
表4：发现队列肿瘤自身抗体组合不同分期的检测敏感性
分期 患者数目 阳性数目 敏感性(％)
T0 4 2 50
T1 8 7 88
T2 8 5 63
T3 6 3 50
未知 15 12 80
实施例2
验证队列包括从上海市肿瘤医院新收集的129例食管癌患者(118例鱗癌、6例低分化癌、2例腺癌、3例神经内分泌癌)，和从上海市肿瘤医院和体检中心收集的88例体检人群，进行抗体标志物的验证。患者信息如表5显示。
表5：验证队列患者特征表
Figure PCTCN2021125167-appb-000002
Figure PCTCN2021125167-appb-000003
针对抗原Trim21，CIP2A，CTAG2，CCDC110，CAGE，MAGEA4，RALA和PDE4DIP，每个肿瘤自身抗体在肿瘤组和对照组的水平分布用散点图来表示(见图1)。肿瘤自身抗体在肿瘤组和对照组的抗体水平分布使用Mann Whitney test进行统计分析，发现针对肿瘤抗原CTAG2、Trim21和RALA的自身抗体在肿瘤组和对照组的水平分布有显著不同(p<0.05)；而针对肿瘤抗原CIP2A、CCDC110、CAGE、MAGEA4和PDE4DIP的自身抗体在肿瘤组和对照组的水平分布没有显著不同。但如果分析在肿瘤组和对照组中大于cutoff值的自身抗体标志物水平在两组人群中的分布，它们有非常明显的不同。正因为如此，所有这些大于cutoff值的阳性标志物对高特异性检测食管癌肿瘤患者有重要意义。
验证队列129例食管癌血清和88例健康对照血清8个肿瘤自身抗体标志物的检测敏感性和特异性如表6所示。单个标志物的特异性都在96.6％或以上，敏感性在7.0～17.1％之间。
表6：验证队列单个自身抗体的检测敏感性和特异性
抗原 敏感性(％) 特异性(％)
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
从8个抗原中选择不同抗原，形成不同抗原组合，由这些不同抗原组合进行对应的肿瘤自身抗体组合的检测，结果在表7显示。可以看出，随着抗原的数目增多，检测敏感性不断增加，而特异性逐渐减少。三个自身抗体组合(抗CCDC110、MAGEA4和PDE4DIP)检测敏感性和特异性分别是41.5％和93.6％，已经达到了相当高的水平。在这三个自身抗体组合基础上增加一个自身抗体，如抗CTAG2，检测敏感性增加到53.6％，特异性仍保持不变。进而，继续增加不同的自身抗体标志物，所形成的自身抗体组合的敏感性更高，而特异性仍保持在90％左右或甚至更高。例如，在这个基础上增加Trim21和RALA检测的敏感性和特异性分别是65.8％和89.3％；而包括针对8个肿瘤抗原Trim21、CIP2A、CTAG2、CCDC110、CAGE、MAGEA4、RALA和PDE4DIP的自身抗体标志物组合在验证队列中的敏感性和特异性分别是70.7％和87.2％。
表7：验证队列不同肿瘤抗原组合检测自身抗体标志物的敏感性和特异性
Figure PCTCN2021125167-appb-000004
实施例3
选择针对8个肿瘤抗原Trim21、CIP2A、CTAG2、CCDC110、CAGE、MAGEA4、RALA和PDE4DIP的自身抗体标志物组合，进行不同分期和分型食管癌的敏感性分析。
参见实施例2，验证队列包括129例食管癌患者(118例鱗癌、6例低分化癌、2例腺癌、3例神经内分泌癌)。验证队列129例食管癌患者包括3例T0期，25例T1期，检测的敏感性分别为0％和32％；18例T2期和58例T3期，敏感性分别是72％和60％；剩下的25例没有分期的信息，敏感性为60％。结果见表8。
表8：验证队列自身抗体组合的不同分期的敏感性
肿瘤分期 患者数目 敏感性(％)
T0 3 0
T1 25 32
T2 18 72
T3 58 60
未知 25 60
验证队列大部分是鱗癌，为118例。虽然西方发达国家食管癌中鳞癌发生率下降，腺癌发生率增加，在当前中国食管癌患者中大部分还是鱗癌。由检测结果可知，本发明优选的8个自身抗体的组合对鳞癌的检测敏感性达到了53％。此外，验证队列中包括2例腺癌，3例神经内分泌癌，自身抗体组合的阳性检出率都为100％。还有6例低分化癌，它的检测敏感性为67％。结果见表9。
表9：验证队列自身抗体组合的不同亚型的敏感性
肿瘤亚型 患者数目 敏感性(％)
腺癌 2 100
神经内分泌癌 3 100
低分化癌 6 67
鱗癌 118 53
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (13)

  1. 一种食管癌的生物标志物，所述生物标志物为自身抗体组合，其包括分别抗以下肿瘤抗原的自身抗体中的至少三个：Trim21、CIP2A、CTAG2、CCDC110、CAGE、MAGEA4、RALA和PDE4DIP。
  2. 根据权利要求1所述的生物标志物，其特征在于，所述自身抗体组合包括分别抗以下肿瘤抗原的自身抗体：CCDC110、MAGEA4和PDE4DIP；
    优选地，所述自身抗体组合还包括分别抗以下肿瘤抗原的自身抗体中的至少一个、至少两个、至少三个、至少四个或甚至至少五个：CTAG2、Trim21、CIP2A、CAGE和RALA；
    更优选地，所述自身抗体组合包括抗以下肿瘤抗原的自身抗体：CTAG2；
    进一步优选地，所述自身抗体组合还包括抗以下肿瘤抗原的自身抗体：Trim21和RALA；和/或，CIP2A和CAGE。
  3. 根据权利要求1或2所述的生物标志物，其特征在于，所述自身抗体组合包括分别抗以下肿瘤抗原的自身抗体：
    (1)CCDC110、MAGEA4和PDE4DIP；
    (2)CCDC110、CTAG2、MAGEA4和PDE4DIP；
    (3)CCDC110、CTAG2、MAGEA4、PDE4DIP、Trim21和RALA；
    (4)CCDC110、CTAG2、MAGEA4、PDE4DIP、CIP2A和CAGE；
    (5)CCDC110、CTAG2、MAGEA4、PDE4DIP、Trim21、RALA、CIP2A和CAGE。
  4. 根据权利要求1至3中任一项所述的生物标志物，其特征在于，所述自身抗体为受试者的全血、血清、血浆、组织或细胞、组织间隙液、脑脊液或尿液中的自身抗体；其中优选地，所述组织或细胞为食管组织或细胞、食管癌组织或细胞或者食管癌的癌旁组织或细胞；
    优选地，所述受试者为哺乳动物，优选为灵长类哺乳动物，更优选为人；
    优选地，所述自身抗体为IgA、IgM或IgG；
    优选地，所述食管癌包括食管鳞癌、食管低分化癌、食管腺癌和食管神经内分泌癌；或者，所述食管癌包括0期、I期、II期、III期。
  5. 一种用于检测如权利要求1至4中任一项所述的生物标志物的试剂。
  6. 根据权利要求5所述的试剂，其特征在于，所述试剂是用于酶联免疫吸附法(ELISA)、蛋白/肽段芯片检测、免疫印迹、微珠免疫检测或微流控 免疫检测的试剂；
    优选地，所述试剂用于通过抗原抗体反应对所述生物标志物进行检测，例如通过ELISA或荧光或化学发光免疫检测。
  7. 根据权利要求5或6所述的试剂，其特征在于，所述试剂为抗原蛋白组合，其包括以下肿瘤抗原中的至少三个：Trim21、CIP2A、CTAG2、CCDC110、CAGE、MAGEA4、RALA和PDE4DIP；
    优选地，所述抗原蛋白组合包括以下肿瘤抗原：CCDC110、MAGEA4和PDE4DIP；
    更优选地，所述抗原蛋白组合还包括以下肿瘤抗原中的至少一个、至少两个、至少三个、至少四个或甚至至少五个：CTAG2、Trim21、CIP2A、CAGE和RALA；
    更优选地，所述抗原蛋白组合包括以下肿瘤抗原：CTAG2；
    进一步优选地，所述自身抗体组合还包括抗以下肿瘤抗原的自身抗体：Trim21和RALA；和/或，CIP2A和CAGE；
    进一步优选地，所述抗原蛋白组合包括以下肿瘤抗原：
    (1)CCDC110、MAGEA4和PDE4DIP；
    (2)CCDC110、CTAG2、MAGEA4和PDE4DIP；
    (3)CCDC110、CTAG2、MAGEA4、PDE4DIP、Trim21和RALA；
    (4)CCDC110、CTAG2、MAGEA4、PDE4DIP、CIP2A和CAGE；
    (5)CCDC110、CTAG2、MAGEA4、PDE4DIP、Trim21、RALA、CIP2A和CAGE。
  8. 如权利要求1至4中任一项所述的生物标志物或如权利要求5至7中任一项所述的试剂在制备用于食管癌的患病风险预测、筛查、预后评估、治疗效果监测或复发检测的产品中的用途。
  9. 根据权利要求8所述的用途，其特征在于，所述食管癌包括食管鳞癌、食管低分化癌、食管腺癌和食管神经内分泌癌；或者，所述食管癌包括0期、I期、II期、III期食管癌；
    优选地，所述产品为试剂盒；
    更优选地，所述试剂盒是用于酶联免疫吸附法(ELISA)、蛋白/肽段芯片检测、免疫印迹、微珠免疫检测或微流控免疫检测的试剂盒；优选地，所述试剂盒用于通过抗原抗体反应对所述生物标志物进行检测，例如为ELISA试剂盒或荧光或化学发光免疫检测试剂盒。
  10. 一种试剂盒，其包含权利要求5至7中任一项所述的试剂。
  11. 根据权利要求10所述的试剂盒，其特征在于，所述试剂盒是用于酶联免疫吸附法(ELISA)、蛋白/肽段芯片检测、免疫印迹、微珠免疫检测或微流控免疫检测的试剂盒；优选地，所述试剂盒用于通过抗原抗体反应对所述生物标志物进行检测，例如为ELISA试剂盒或荧光或化学发光免疫检测试剂盒。
  12. 一种食管癌的患病风险预测、筛查、预后评估、治疗效果监测或复发监测的方法，其包括以下步骤：
    (1)对来自受试者的样本进行权利要求1至4中任一项所述的自身抗体组合中的每个自身抗体的定量；
    (2)将所述自身抗体的量与参考阈值进行比较，在其高于参考阈值时，确定所述受试者具有食管癌患病风险、患有食管癌、预后不良或食管癌治疗效果差。
  13. 根据权利要求12所述的方法，其特征在于，在步骤(1)中，所述定量包括采用权利要求5至7中任一项所述的试剂或包含所述试剂的试剂盒对对所述自身抗体组合中的每个自身抗体进行检测；所述受试者为哺乳动物，优选为灵长类哺乳动物，更优选为人；
    优选地，所述食管癌包括食管鳞癌、食管低分化癌、食管腺癌和食管神经内分泌癌；或者，所述食管癌包括0期、I期、II期、III期食管癌；
    优选地，所述样本为受试者的全血、血清、血浆、组织或细胞、组织间隙液、脑脊液或尿液；其中优选地，所述组织或细胞为食管组织或细胞、食管癌组织或细胞或者食管癌的癌旁组织或细胞；
    优选地，在步骤(2)中，所述参考阈值是来自健康人或健康人群的参照水平。
PCT/CN2021/125167 2020-10-21 2021-10-21 食管癌的生物标志物及其应用 WO2022083673A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011130430.7 2020-10-21
CN202011130430.7A CN112362871B (zh) 2020-10-21 2020-10-21 食管癌的生物标志物及其应用

Publications (1)

Publication Number Publication Date
WO2022083673A1 true WO2022083673A1 (zh) 2022-04-28

Family

ID=74511357

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/125167 WO2022083673A1 (zh) 2020-10-21 2021-10-21 食管癌的生物标志物及其应用

Country Status (2)

Country Link
CN (1) CN112362871B (zh)
WO (1) WO2022083673A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114924075A (zh) * 2022-05-26 2022-08-19 郑州大学第一附属医院 基于聚焦阵列蛋白芯片筛选用于贲门腺癌诊断的生物标志物及其应用
CN116121377A (zh) * 2022-11-01 2023-05-16 山西医科大学 一种食管鳞癌外泌体富含的miRNA作为诊断食管鳞癌的标志物中的应用
CN116298289A (zh) * 2023-01-30 2023-06-23 上海秤信生物科技有限公司 一种预测肺癌免疫新辅助治疗效果的生物标志物及其应用
CN116622849A (zh) * 2023-05-31 2023-08-22 山东大学齐鲁医院 circ0337-122aa的检测试剂作为食管鳞癌预后试剂的应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112362871B (zh) * 2020-10-21 2023-06-06 杭州凯保罗生物科技有限公司 食管癌的生物标志物及其应用
CN113671180B (zh) * 2021-09-16 2023-07-07 郑州大学 Paip1自身抗体在食管鳞癌辅助诊断中的应用
CN114167059B (zh) * 2021-11-03 2023-07-14 郑州大学 一种用于食管鳞癌诊断的生物标志物及检测试剂盒
CN115372616B (zh) * 2022-07-15 2023-09-15 杭州凯保罗生物科技有限公司 胃癌相关的生物标志物及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030232399A1 (en) * 2000-06-14 2003-12-18 Robertson John Forsyth Russell Cancer detection methods and reagents
CN101435822A (zh) * 2008-11-05 2009-05-20 广州市搏克生物技术有限公司 一种血清自身抗体用于食管鳞癌诊断的方法
US20100047830A1 (en) * 2007-02-01 2010-02-25 University Of Florida Research Foundation, Inc. Compositions and methods for detecting cancers in a subject
US20100204055A1 (en) * 2008-12-05 2010-08-12 Bonner-Ferraby Phoebe W Autoantibody detection systems and methods
CN103869086A (zh) * 2014-04-14 2014-06-18 杭州凯保罗生物科技有限公司 一种血清自身抗体检测试剂盒
WO2017121622A1 (en) * 2016-01-14 2017-07-20 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Serological autoantibodies as biomarker for colorectal cancer
CN112362871A (zh) * 2020-10-21 2021-02-12 杭州凯保罗生物科技有限公司 食管癌的生物标志物及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008130568A1 (en) * 2007-04-16 2008-10-30 Oncomed Pharmaceuticals, Inc. Compositions and methods for treating and diagnosing cancer
US20150198600A1 (en) * 2012-07-27 2015-07-16 Rowan University Autoantibody Profiles in the Early Detection and Diagnosis of Cancer
WO2014193999A2 (en) * 2013-05-28 2014-12-04 Caris Science, Inc. Biomarker methods and compositions
US11446398B2 (en) * 2016-04-11 2022-09-20 Obsidian Therapeutics, Inc. Regulated biocircuit systems
CN109342727B (zh) * 2018-10-15 2021-11-12 汕头大学医学院附属肿瘤医院 食管鳞状细胞癌自身抗体分子标志物模型及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030232399A1 (en) * 2000-06-14 2003-12-18 Robertson John Forsyth Russell Cancer detection methods and reagents
US20100047830A1 (en) * 2007-02-01 2010-02-25 University Of Florida Research Foundation, Inc. Compositions and methods for detecting cancers in a subject
CN101435822A (zh) * 2008-11-05 2009-05-20 广州市搏克生物技术有限公司 一种血清自身抗体用于食管鳞癌诊断的方法
US20100204055A1 (en) * 2008-12-05 2010-08-12 Bonner-Ferraby Phoebe W Autoantibody detection systems and methods
CN103869086A (zh) * 2014-04-14 2014-06-18 杭州凯保罗生物科技有限公司 一种血清自身抗体检测试剂盒
WO2017121622A1 (en) * 2016-01-14 2017-07-20 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Serological autoantibodies as biomarker for colorectal cancer
CN112362871A (zh) * 2020-10-21 2021-02-12 杭州凯保罗生物科技有限公司 食管癌的生物标志物及其应用

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114924075A (zh) * 2022-05-26 2022-08-19 郑州大学第一附属医院 基于聚焦阵列蛋白芯片筛选用于贲门腺癌诊断的生物标志物及其应用
CN116121377A (zh) * 2022-11-01 2023-05-16 山西医科大学 一种食管鳞癌外泌体富含的miRNA作为诊断食管鳞癌的标志物中的应用
CN116298289A (zh) * 2023-01-30 2023-06-23 上海秤信生物科技有限公司 一种预测肺癌免疫新辅助治疗效果的生物标志物及其应用
CN116298289B (zh) * 2023-01-30 2023-11-03 上海秤信生物科技有限公司 一种预测肺癌免疫新辅助治疗效果的生物标志物及其应用
CN116622849A (zh) * 2023-05-31 2023-08-22 山东大学齐鲁医院 circ0337-122aa的检测试剂作为食管鳞癌预后试剂的应用
CN116622849B (zh) * 2023-05-31 2024-04-23 山东大学齐鲁医院 circ0337-122aa的检测试剂作为食管鳞癌预后试剂的应用

Also Published As

Publication number Publication date
CN112362871A (zh) 2021-02-12
CN112362871B (zh) 2023-06-06

Similar Documents

Publication Publication Date Title
WO2022083673A1 (zh) 食管癌的生物标志物及其应用
WO2022063156A1 (zh) 乳腺癌的生物标志物及其应用
JP2020515826A (ja) 抗体アッセイ
US20140271621A1 (en) Methods of prognosis and diagnosis of pancreatic cancer
CN115877006B (zh) 卵巢癌相关的生物标志物及其应用
ES2684976T3 (es) Biomarcadores maternos para diabetes gestacional
EP2414843A1 (en) Detection of fibrin and fibrinogen degradation products and associated methods of production and use for the detection and monitoring of cancer
WO2016195051A1 (ja) 膵がんを診断するための血漿バイオマーカーパネル
AU2010252907B2 (en) Methods for the diagnosis or prognosis of colorectal cancer
CN109116023B (zh) 一种肺癌标志物抗-mmp12自身抗体及其应用
CN115372616B (zh) 胃癌相关的生物标志物及其应用
JP6876301B2 (ja) 肝細胞がん患者の再発リスク予測を補助する方法、装置、コンピュータプログラム製品及びキット
WO2013119279A2 (en) Assays and methods for the diagnosis of ovarian cancer
CN116413430B (zh) 一种用于肝癌早期预测的自身抗体/抗原组合及检测试剂盒
US8697368B2 (en) Diagnostic marker for lung cancer comprising HPαR as active ingredient
KR102018209B1 (ko) 위암 진단용 조성물 및 상기 조성물을 이용한 위암 진단 방법
US20230375550A1 (en) Method for diagnosing breast cancer by using biomarker
CN116381237B (zh) 一种甲状腺癌早期预测系统及其应用
US10866239B2 (en) Methods for colon hyperproliferative disorder detection, prognosis, and diagnosis
CN116539885B (zh) 用于乳腺癌早期检测的肿瘤自身抗原/抗体组合及应用
CN116298295B (zh) 用于结直肠癌早期检测的肿瘤自身抗原/抗体组合及应用
WO2023230584A1 (en) Methods and systems for risk stratification and management of bladder cancer
CN114167059A (zh) 一种用于食管鳞癌诊断的生物标志物及检测试剂盒
TW201928351A (zh) 抗體測定
WO2017060840A1 (en) Use of htra1 for early detection of colorectal cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21882088

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21882088

Country of ref document: EP

Kind code of ref document: A1