A chest tumor diagnostic marker CPD and its related diagnostic products and applications

Abstract

The present invention discloses a chest tumor diagnostic marker CPD and related diagnostic products and applications thereof. The present invention discovers for the first time that CPD can be used for the early diagnosis of esophageal cancer. Verification through real clinical samples collected by the present invention shows that the CPD has good diagnostic efficacy for the diagnosis of esophageal cancer, and has high accuracy, sensitivity and specificity. The present invention provides new ideas and strategies for the technical field of esophageal cancer diagnosis, and has important scientific significance and clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically, relates to a chest tumor diagnostic marker, and more specifically, to a chest tumor diagnostic marker CPD and related diagnostic products and applications thereof. Background Art

[0002] Esophageal cancer is one of the common malignant tumors. According to global cancer statistics, the incidence of esophageal cancer ranks 7th and the mortality rate ranks 6th. The most common histological subtypes are squamous cell carcinoma and adenocarcinoma, of which squamous cell carcinoma accounts for more than 90%, and their biological characteristics are different. Precision treatment is increasingly becoming the mainstream of esophageal cancer treatment. Among them, the TNM staging based on tumor size, lymph node metastasis and distant metastasis, combined with esophageal endoscopy and pathological parameters to form a comprehensive clinical evaluation system for esophageal cancer, is the main reference standard for esophageal cancer treatment and has promoted the improvement of esophageal cancer treatment. In short, a clinical treatment framework has been formed for radical surgical resection of early esophageal cancer, postoperative adjuvant radiotherapy and chemotherapy for advanced and late esophageal cancer, and comprehensive treatment for advanced and metastatic esophageal cancer.

[0003] With the progress of research in genomics, tumor biology, and tumor immunology, targeted therapy for esophageal cancer driver genes and immune checkpoint therapy for immune characteristics have shown better tumor remission, improved prognosis, and improved quality of life, compared with traditional cytotoxic chemotherapy, both in the neoadjuvant / adjuvant treatment of early esophageal cancer and in the comprehensive treatment of advanced esophageal cancer, while showing lower toxic side effects. However, esophageal cancer is a highly heterogeneous tumor, and its origin, molecular characteristics, and unique tumor microenvironment will affect the clinical treatment effect. At the same time, chemotherapy, targeted therapy, and immunotherapy all have problems of insensitivity and drug resistance. Therefore, the precise classification of esophageal cancer and the research on new targets for clinical prevention and treatment still need to be deepened, and key genes are an important direction for exploring the mechanism of occurrence and development of esophageal cancer and even precise classification and clinical prevention and treatment.

[0004] Carboxypeptidase-D (CPD) regulates the activity of key proteins through post-translational modification and participates in the regulation of various life processes. In recent years, it has been found that CPD is closely related to the occurrence and development of various tumors, but research on CPD in esophageal cancer is still lacking. This study explored the expression characteristics, clinical significance and biological functions of CPD in esophageal cancer, providing some inspiration for the study of the carcinogenic mechanism of esophageal cancer and even the exploration of new targets for precision prevention and treatment. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a chest tumor diagnostic marker CPD and its related diagnostic products and applications.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] The first aspect of the present invention provides the use of a reagent for detecting the expression level of the biomarker CPD in a sample in the preparation of a product for diagnosing or identifying esophageal cancer.

[0008] Further, the reagent includes:

[0009] Reagents for detecting the DNA level of CPD in a sample;

[0010] Reagents for detecting RNA levels of CPD in a sample;

[0011] A reagent for detecting the protein expression level of CPD in a sample; or

[0012] A reagent for detecting the number of CPD-positive cells in a sample.

[0013] Further, the reagents for detecting the DNA level of CPD in the sample include reagents for detecting the DNA expression level, DNA methylation level and / or DNA phosphorylation level of CPD in the sample;

[0014] Optionally, the detecting the DNA level of CPD in the sample comprises a reagent detecting the DNA level by sequencing technology;

[0015] Optionally, the reagent for detecting the RNA level of CPD in the sample includes a reagent for detecting the mRNA, lncRNA and / or miRNA expression level of CPD in the sample;

[0016] Optionally, the reagent for detecting the RNA level of CPD in the sample includes primers that specifically amplify CPD and / or probes that specifically recognize CPD.

[0017] Further, the reagent for detecting the protein expression level of CPD in the sample includes an affinity protein that specifically binds to the protein encoded by CPD;

[0018] Optionally, the reagent for detecting the number of CPD-positive expressing cells in a sample includes a reagent for detecting the number of CPD-positive expressing cells by immunohistochemistry;

[0019] Optionally, the affinity protein includes an antibody, an antibody functional fragment, an agglutinating agent, a receptor and / or a coupled antibody that specifically binds to a protein encoded by a CPD.

[0020] Furthermore, the esophageal cancer includes esophageal squamous cell carcinoma and / or esophageal adenocarcinoma;

[0021] Optionally, the diagnosing esophageal cancer includes diagnosing esophageal squamous cell carcinoma and / or diagnosing esophageal adenocarcinoma;

[0022] Optionally, identifying esophageal cancer includes identifying and distinguishing between esophageal squamous cell carcinoma and esophageal adenocarcinoma.

[0023] Optionally, the identifying of esophageal cancer further includes identifying and distinguishing any two or three of esophageal squamous cell carcinoma, esophageal adenocarcinoma and Barrett's esophagus.

[0024] Furthermore, the sample includes a tissue sample, a blood sample, a serum sample, a plasma sample, a cell sample, a urine sample and / or an exosome sample derived from a subject.

[0025] In the present invention, the CPD refers to carboxypeptidase-D (CPD), the full name of which is carboxypeptidase D [Homo sapiens (human)], and its Gene ID in NCBI is 1362, and its detailed information can be obtained in the NCBI database (https: / / www.ncbi.nlm.nih.gov / gene / ) based on the above Gene ID. In the present invention, the CPD includes CPD gene and CPD protein.

[0026] In some embodiments, the CPD has a sequence known in the art or a derivative thereof. In some embodiments, the CPD is a molecule comprising the following sequence: (a) a CPD molecule having a sequence such as Gene ID: 1362 (human), Gene ID: 12874 (rat), Gene ID: 25306 (Norway rat), Gene ID: 480636 (dog), Gene ID: 101345198 (cattle); (b) a molecule hybridizing with a sequence defined in (a) under stringent conditions; (c) a molecule having a sequence homology of more than 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more, or any value or range of values ​​therebetween) with the sequence of the molecule shown in (a) or (b), such as a CPD molecule obtained by codon optimization.

[0027] In some embodiments, the sample includes any sample (subject source) collected and sampled from a cell, tissue or body fluid, wherein the sample includes but is not limited to: the source of the tissue or cell sample can be from a fresh, frozen and / or preserved solid tissue of an organ or tissue sample, or a biopsy or aspirate, blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid. Tissue samples can be primary or in vitro cultured cells or cell lines. Optionally, tissue or cell samples are obtained from diseased tissues / organs. Tissue samples may contain compounds naturally mixed with the tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or similar compounds. In some embodiments, the sample to be tested is tissue or blood derived from a subject.

[0028] In some embodiments, the sample includes, but is not limited to, tissue, blood, tissue-derived cells, blood-derived cells, serum, plasma, lymph, synovial fluid, exosomes, cell extracts, feces, urine, saliva, sputum, joint cavity fluid, pleural effusion, serous cavity effusion, lymph, cerebrospinal fluid, uterine cavity fluid, digestive fluid, bile, alveolar bronchial washings, organs, and any combination thereof. In a preferred embodiment, the sample is selected from the tissue of the subject.

[0029] In the present invention, the biomarker refers to a gene differentially expressed between a subject with a second phenotype (e.g., no disease) and a subject with a first phenotype (e.g., with a disease), specifically, compared with a biological sample from a subject with a second phenotype (e.g., no disease) or a group of subjects, it is significantly differentially present (i.e., increased or decreased) in a biological sample from a subject with a first phenotype (e.g., with a disease) or a group of subjects; the biomarker may be differentially present at any level. In a specific embodiment of the present invention, the biomarker refers to CPD.

[0030] In the present invention, it is verified by real clinical samples collected by the present invention that the CPD has good diagnostic efficacy for esophageal cancer, wherein the diagnostic efficacy is verified by a receiver operating characteristic (ROC) curve, and the area under the curve (AUC) refers to the area under the ROC curve well known to those skilled in the art. The determination of the area under the curve (AUC) helps to compare the accuracy of the classifier across the overall data range.

[0031] A classifier with a larger area under the curve (AUC) has a greater ability to accurately classify unknowns between two groups of interest (e.g., cancer samples and normal or control samples). In terms of distinguishing two populations (e.g., esophageal cancer group and non-esophageal cancer control group), the receiver operating characteristic curve (ROC) is useful for graphically representing the performance of a specific feature (e.g., any item of biomarkers and / or additional biomedical information described in the present invention). Typically, based on a single feature value, the above-mentioned feature data of the entire population (e.g., patient group and control group) are arranged in ascending order. Then, for each value of the above-mentioned feature, the true positive rate and false positive rate for the data are calculated. The above-mentioned true positive rate is determined by dividing the total number of cases by calculating the number of cases above the value for its characteristic. The above-mentioned false positive rate is determined by dividing the total number of controls by calculating the number of controls above the value for its characteristic. Although this definition refers to the case where the characteristics of the patient group are high relative to the control group, the definition is also applicable to the case where the characteristics of the patient group are low relative to the control group (in this case, the number of samples with values ​​lower than the above-mentioned characteristics can be calculated).

[0032] The receiver operating characteristic curve (ROC) can be generated for other single calculations and for single characteristics. For example, two or more characteristics can be mathematically combined (e.g., addition, subtraction, multiplication, etc.) to provide a single sum value, which can be represented by the receiver operating characteristic curve (ROC). Additionally, the combination of multiple characteristics that can derive a single calculation value can be drawn with the receiver operating characteristic curve (ROC). These characteristic combinations can constitute a test. The above-mentioned receiver operating characteristic curve (ROC) is a graph representing the true positive rate (sensitivity) of the test relative to the false positive rate (1-specificity) of the test.

[0033] In some embodiments, the sequencing technology refers to any reagent that can be used to detect the DNA expression level, DNA methylation level, DNA acetylation level, DNA phosphorylation level, DNA hydroxymethylation level and / or SNP site genotype of CPD in a sample. Exemplarily, the sequencing technology includes but is not limited to: Sanger sequencing technology, Illumina sequencing technology, Ion Torrent sequencing technology, PacBio sequencing technology, Oxford Nanopore long-read sequencing technology.

[0034] In some embodiments, the reagent can detect the expression level of CPD in a test sample derived from a subject through spatial transcriptomics analysis (Digital Spatial Profiler, DSP) technology, sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology and / or protein immunoassay technology.

[0035] In the present invention, the diagnosis or identification refers to the discovery, judgment or recognition of the health status or condition of an individual based on one or more symptoms, data or other information related to the individual. The health status of an individual can be diagnosed as healthy / normal (i.e., the absence of a disease or illness), or can be diagnosed as unhealthy / abnormal (i.e., the presence of a disease or illness), or can be diagnosed as a specific severity or degree of progression of the disease. The term diagnosis or identification includes the early discovery of diseases / disorders associated with specific diseases or illnesses; the characteristics or classification of the disease; the discovery of the progression, cure or recurrence of the disease; and the discovery of the individual's response to the disease after treatment or treatment.

[0036] A second aspect of the present invention provides a product for diagnosing or identifying esophageal cancer.

[0037] Furthermore, the product comprises the reagent for detecting the expression level of the biomarker CPD in a sample as described in the first aspect of the present invention.

[0038] Further, the product includes a detection kit, a detection chip and / or a detection test strip;

[0039] Optionally, the detection kit comprises primers, probes or chips that specifically bind to the biomarker CPD;

[0040] Optionally, the detection chip includes a solid phase carrier and a probe attached to the solid phase carrier that specifically recognizes the biomarker CPD.

[0041] In the present invention, the primer refers to a 7-50 nucleic acid sequence that can form a base pair complementary to the template strand and acts as a starting point for replicating the template strand. Primers are usually synthesized, but naturally occurring nucleic acids can also be used. The sequence of the primer does not necessarily need to be exactly the same as the sequence of the template, as long as it is sufficiently complementary and can hybridize with the template.

[0042] In the present invention, the probe refers to a nucleic acid fragment as short as a few to as long as hundreds of bases, such as RNA or DNA, which can establish specific binding with mRNA and can determine the presence of specific mRNA due to labeling. The probe can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, and RNA probes.

[0043] In some embodiments, the detection kit further comprises: a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant or a solvent, such as a solution for suspending or fixing cells, a detectable label or marker, a solution for facilitating nucleic acid hybridization, a solution for lysing cells, or a solution for nucleic acid purification.

[0044] In some embodiments, the detection kit may also be accompanied by instructions for use of the detection kit, which record how to use the kit for detection and how to use the test results to determine whether the subject has esophageal cancer, the type of esophageal cancer, or the risk of esophageal cancer.

[0045] In some embodiments, the detection kit includes, but is not limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid detection kit, or an MRM (multiple reaction monitoring) kit.

[0046] In some embodiments, the RT-PCR kit may further include the elements necessary for reverse transcription polymerase chain reaction. The RT-PCR kit includes a pair of primers specific for the gene encoding the marker protein. Each primer is a nucleotide with a specific nucleotide sequence for the gene. The RT-PCR kit may also include a test tube or a suitable vessel, a reaction buffer (different pH values ​​and magnesium concentrations), a deoxynucleotide (dNTP), an enzyme (such as Taq polymerase and reverse transcriptase), a deoxyribonuclease inhibitor, a ribonuclease inhibitor, DEPC-water and sterile water.

[0047] In some embodiments, the ELISA kit may further include elements necessary for performing ELISA. The ELISA kit may include antibodies specific for protein (CPD protein of the present invention). The antibody has high selectivity and affinity for marker protein, no cross-reactivity with other proteins, and may be a monoclonal antibody, a polyclonal antibody or a recombinant antibody. In addition, the ELISA kit may include antibodies specific for control proteins. In addition, the ELISA kit may further include reagents capable of detecting the bound antibody, for example, a labeled second antibody, a chromophore, an enzyme (e.g., conjugated with an antibody), and a substrate thereof or a substance capable of binding the antibody.

[0048] In some embodiments, the detection kit of the present invention can be used to detect the CPD expression level by various methods selected from the group consisting of, but not limited to, real-time quantitative reverse transcription PCR, biochip detection, Southern blotting, Northern blotting or in situ hybridization. A person of ordinary skill in the art can adjust and change the detection mode or detection method according to actual conditions and needs.

[0049] In some embodiments, the solid phase carrier for preparing the detection chip described in the present invention includes various commonly used materials in the field of gene chips, such as (but not limited to): nylon membranes, glass slides or silicon wafers modified with active groups (such as aldehyde groups, amino groups, etc.), unmodified glass slides, plastic sheets, etc.

[0050] In some embodiments, the detection chip (microarray chip) refers to a solid support comprising a nucleic acid or peptide probe connected. The array usually comprises a plurality of different nucleic acids or peptide probes connected to the substrate surface according to different known positions. These arrays, also referred to as microarrays, can usually be produced using mechanical synthesis methods or light-guided synthesis methods, which incorporate a combination of photolithography and solid phase synthesis methods. The array can include a flat surface, or can be a bead, gel, polymer surface, such as a fiber of an optical fiber, glass or any other suitable substrate nucleic acid or peptide. The array can be packaged in a certain manner to allow the diagnosis of a fully functional device or other manipulations.

[0051] In some embodiments, the detection chip includes a gene chip and a protein chip; the gene chip includes a solid phase carrier; and oligonucleotide probes fixed in order on the solid phase carrier, and the oligonucleotide probes specifically correspond to part or all of the sequence shown by the CPD. The protein chip includes a solid phase carrier, and specific antibodies or ligands of proteins encoded by the CPD fixed on the solid phase carrier. The solid phase carrier can be made of various commonly used materials in the chip field, such as but not limited to: plastic products, microparticles, membrane carriers, etc.

[0052] The third aspect of the present invention provides the use of a reagent for detecting the expression level of CPD in a sample in the preparation of a system and / or device for diagnosing or identifying esophageal cancer.

[0053] In the present invention, the system and / or device is a method for distinguishing different components, elements, parts, parts or assemblies at different levels. However, if other words can achieve the same purpose, the words can be replaced by other expressions. It is well known to those skilled in the art that the present invention can be implemented as an apparatus, method or computer program product. Therefore, the content disclosed in the present invention can be specifically implemented in the following forms, that is, it can be complete hardware, it can be complete software (including firmware, resident software, microcode, etc.), and it can also be a combination of hardware and software. In addition, in some specific embodiments, the present invention can also be implemented in the form of a computer program product in one or more computer-readable media, and the computer-readable medium contains a computer-readable program code.

[0054] A fourth aspect of the present invention provides a system for diagnosing or identifying esophageal cancer.

[0055] Further, the system includes a processor, an input module, and an output module;

[0056] Among them, the processor is used to perform logical operations on the input information using bioinformatics methods; the input module is used to input the CPD expression level in the subject's sample, and contains a computer-readable medium of instructions, which, when executed by the processor, executes an algorithm on the CPD input expression level; the output module is used to output whether the subject has esophageal cancer, the risk of esophageal cancer, or the type of esophageal cancer.

[0057] The present invention also provides a method for diagnosing or identifying esophageal cancer, the method comprising the following steps:

[0058] (1) Collect samples from subjects;

[0059] (2) detecting the expression level of CPD in samples from subjects;

[0060] (3) Diagnosing whether the subject has esophageal cancer or the risk of esophageal cancer based on the expression level of the detected CPD, or distinguishing the type of esophageal cancer suffered by the subject.

[0061] The present invention also provides a biomarker for diagnosing or identifying esophageal cancer, wherein the biomarker is CPD.

[0062] The present invention also provides the use of the CPD inhibitor in preparing a medicine for treating and / or preventing esophageal cancer.

[0063] In some embodiments, the CPD inhibitor includes any agent capable of inhibiting CPD expression, including but not limited to: shRNA, siRNA, miRNA, antisense nucleic acid, CRISPR gene editing reagents, small molecule compounds, peptides, peptide mimetics, matrix analogs, aptamers, antibodies, dsRNA, etc.

[0064] In some embodiments, according to the CPD sequence information provided by the present invention, the CPD interfering molecule as described above can be prepared by a preparation method known in the art. In the present invention, there is no particular limitation on the preparation method of the interfering molecule as described above, including but not limited to: chemical synthesis, in vitro transcription, etc. The interfering molecule can be delivered to the target cell by using an appropriate transfection reagent, or can also be delivered to the target cell by using a variety of techniques known in the art.

[0065] In some embodiments, the agent for knocking out the CPD gene or inhibiting the expression of the CPD gene also includes a small molecule compound targeting FOXR1. Those skilled in the art can use conventional screening methods in the art to screen such small molecule compounds, and the small molecule compounds that can knock out the CPD gene or inhibit the expression of the CPD gene obtained by screening are also included in the protection scope of the present invention.

[0066] The present invention also provides a method for treating and / or preventing esophageal cancer, comprising: administering a therapeutically and / or preventively effective amount of the CPD inhibitor as described above to a subject in need thereof.

[0067] In the present invention, the subject refers to any animal, including humans and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human.

[0068] In the present invention, the therapeutically effective amount refers to an amount sufficient to provide a therapeutic benefit in the process of treating a disease, disorder or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition. The therapeutically effective amount of a compound refers to the amount of a therapeutic agent that provides a therapeutic benefit in the process of treating a disease, disorder or condition when used alone or in combination with other therapies.

[0069] In the present invention, the preventive effective amount refers to an amount sufficient to prevent a disease, disorder or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder or condition, or an amount to prevent the recurrence of a disease, disorder or condition. A preventive effective amount of a compound refers to an amount of a therapeutic agent, when used alone or in combination with other agents, that provides a preventive benefit in the process of preventing a disease, disorder or condition.

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] The present invention discovers for the first time that CPD can be used for the early diagnosis of esophageal cancer. Verification using real clinical samples collected by the present invention shows that the CPD has good diagnostic efficacy for esophageal cancer, with high accuracy, sensitivity and specificity. The present invention solves the technical problems of low sensitivity and specificity for esophageal cancer diagnosis in the prior art, and poor patient compliance due to various reasons, and provides new ideas and strategies for the technical field of esophageal cancer diagnosis, which has important scientific significance and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 :Gene expression matrix and clinical information of different esophageal cancer datasets;

[0073] Figure 2: Gene expression characteristics of CPD in esophageal cancer-normal tissues, including: A: Unannotated UMAP map of 8 esophageal cancer datasets; B: UMAP map of the integrated esophageal cancer dataset; CD: Gene expression characteristics of CPD in cancer tissues-normal tissues in esophageal cancer datasets (training set and validation set); EF: ROC curve shows a discriminatory effect between esophageal cancer tissues and normal tissues (training set and validation set);

[0074] Figure 3 : Gene expression characteristics of CPD in esophageal squamous cell carcinoma-normal tissues, where AB figure: Gene expression characteristics of CPD in esophageal squamous cell carcinoma tissue-normal tissues in esophageal cancer data set (training set and validation set); CD figure: ROC curve shows discrimination effect in esophageal squamous cell carcinoma tissue and normal tissue (training set and validation set);

[0075] Figure 4 : Gene expression characteristics of CPD in esophageal adenocarcinoma-normal tissues, where A, C, E: Gene expression characteristics of CPD in esophageal adenocarcinoma-normal tissues, Barrett's esophagus and esophageal adenocarcinoma, Barrett's esophagus and normal tissues in esophageal cancer data set (training set and validation set); B, D, F: ROC curves show discrimination effect in esophageal adenocarcinoma-normal tissues, Barrett's esophagus and esophageal adenocarcinoma, Barrett's esophagus and normal tissues (training set and validation set);

[0076] Figure 5 : Gene expression characteristics of CPD in esophageal adenocarcinoma-esophageal squamous cell carcinoma, where A: In the esophageal cancer dataset (training set and validation set), the gene expression characteristics of CPD in esophageal adenocarcinoma-esophageal squamous cell carcinoma tissues; B: ROC curve shows a discriminatory effect in esophageal adenocarcinoma-esophageal squamous cell carcinoma tissues (training set and validation set);

[0077] Figure 6 : Analysis of biological functions of CPD in esophageal adenocarcinoma and esophageal squamous cell carcinoma, wherein, Figure A: Analysis of biological functions of CPD in esophageal adenocarcinoma; Figure B: Analysis of biological functions of CPD in esophageal squamous cell carcinoma;

[0078] Figure 7 : Analysis of the protein content of CPD in esophageal cancer, where AG: immunohistochemical analysis of CPD in esophageal cancer tissue-normal tissue; H: protein content analysis of CPD in esophageal cancer tissue-normal tissue;

[0079] Figure 8 : The results of tissue chip analysis of CPD in real clinical esophageal cancer tissue samples and adjacent tissue samples collected by the present invention;

[0080] Fig. 9 : Diagram showing the differential expression of CPD in real clinical esophageal cancer tissue samples and adjacent tissue samples collected by the present invention and the result of diagnostic efficacy analysis. DETAILED DESCRIPTION

[0081] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0082] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0083] Example 1 Study on the diagnosis and biological function of CPD in esophageal cancer

[0084] 1. Data and sample sources

[0085] (1) Public dataset information

[0086] The gene expression matrix and clinical information of different esophageal cancer datasets were obtained through the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ) (see Figure 1 ).

[0087] Among them, 8 esophageal cancer datasets were integrated for analyzing gene expression characteristics, including: dataset (number of tumor tissue / normal tissue samples), GSE1420 (16 / 8), GSE20347 (17 / 17), GSE23400 (53 / 53), GSE26886 (50 / 19), GSE38129 (30 / 30), GSE5364 (16 / 13), GSE77861 (7 / 7), GSE92396 (12 / 9).

[0088] (2) Patients and organizations

[0089] Seven cases of esophageal cancer tissues were obtained from the Department of Thoracic Surgery, the Second Affiliated Hospital of the Air Force Medical University, and detailed clinical information was collected, collated, and analyzed. The tissues were divided into two parts, one for paraffin embedding and immunohistochemistry experiments, and one frozen tissue for protein, RNA extraction and subsequent experiments. The use of human pathological specimens involved in this study complies with the ethical requirements of the Ethics Committee of the Second Affiliated Hospital of the Air Force Medical University, and also complies with the 1964 Declaration of Helsinki and its subsequent amendments and supplements. All subjects gave informed consent and signed the informed consent form.

[0090] 2. Experimental methods

[0091] (1) Immunohistochemistry

[0092] After the above-mentioned esophageal cancer and adjacent tissues were collected, they were fixed with 4% paraformaldehyde, dehydrated with gradient ethanol, transparentized with xylene, immersed in wax, and embedded in paraffin. After the paraffin sections were dried, dewaxed with xylene, and hydrated with gradient ethanol, they were washed with phosphate buffer and microwave heated for antigen repair. After 3% hydrogen peroxide treatment of endogenous peroxidase, they were blocked and incubated with primary antibodies overnight (anti-CPD antibody: DF9339, Affinity Biosciences, OH, USA). After rinsing the next day, they were incubated with enzyme-labeled secondary antibodies, washed, and then developed with DAB. The protein content in immunohistochemistry was measured by semi-quantitative histochemical method: H-score = Σ(pi*i), "pi" represents the percentage of positive cells, and "I" represents the signal intensity.

[0093] (2) Western-blot (WB)

[0094] The frozen tissue was lysed with RIPA lysis buffer containing phosphatase and protease inhibitors, and the lysate was added with loading buffer containing sodium dodecyl sulfate (SDS) and boiled in boiling water, and then separated by SDS-polyacrylamide gel (PAGE) electrophoresis. The protein was then transferred to a nitrocellulose membrane. After blocking with 5% skim milk, it was incubated with a specific primary antibody overnight (CPD, PA5-103707, Invitrogen, MA, USA), incubated with an enzyme-labeled secondary antibody of the corresponding species, and detected by an ECL kit after washing.

[0095] 3. Methods and statistical analysis

[0096] Gene expression data were standardized by Z-scaling. The Wilcoxon test was used to evaluate the gene expression and protein expression of CPD in esophageal cancer tissues and normal tissues. The high and low CPD expression patient groups in esophageal cancer data were classified by median, the correlation between CPD and genes was evaluated by Pearson correlation analysis, and the biological enrichment of CPD was evaluated in the hallmark database using the geneset enrichment analysis (GSEA). R language was used for graphics drawing and statistical analysis (statistically significant: two-sided p < 0.05).

[0097] 4. Experimental results

[0098] (1) CPD in esophageal cancer shows high gene expression in cancer

[0099] Since the sample size of both normal tissue and esophageal cancer tissue in the above single data set is small, no statistically significant comparison can be made. Therefore, this example integrates the above gene set. It can be seen that there is an obvious batch effect before annotation ( Figure 2 A), the batch effect is significantly weakened after integration (through U-map, Figure 2 B). We divided the dataset into training and validation sets in a 6:4 ratio according to the ratio of esophageal cancer to normal tissue (Normal tissue / Tumor tissue, 156 / 173). We can see that in both the validation and training sets, the CPD gene in esophageal cancer tissues is significantly more highly expressed than in normal tissues ( Figure 2 C, training set; Figure 2 D, validation set). At the same time, the ROC curve shows a significant discrimination effect ( Figure 2 E, training set; Figure 2 F, validation set). The above results show that CPD has good diagnostic efficacy for esophageal cancer (AUC value is as high as 0.800).

[0100] (2) CPD in esophageal squamous cell carcinoma shows high gene expression in cancer

[0101] Esophageal squamous cell carcinoma is an important pathological subtype of esophageal cancer. In this example, according to the ratio of esophageal squamous cell carcinoma to normal tissue (Normal tissue / squamous cell carcinoma, 156 / 116) of the overall data set, a 6:4 stratification is performed to divide the data set into a training set and a validation set. It can be seen that in both the validation set and the training set, the CPD gene in the tissue is significantly overexpressed in esophageal squamous cell carcinoma tissue compared with normal tissue ( Figure 3 A, training set; Figure 3B, validation set). At the same time, the ROC curve shows a significant discrimination effect ( Figure 3 C, training set; Figure 3 D, validation set). The above results show that CPD has good diagnostic efficacy for esophageal squamous cell carcinoma (AUC value is as high as 0.877).

[0102] (3) Analysis of CPD gene expression in esophageal adenocarcinoma and normal tissues

[0103] Due to the small number of cases of esophageal adenocarcinoma, cross-validation cannot be performed. This example uniformly focuses on the overall data set to explore expression analysis. Esophageal adenocarcinoma follows the transition from normal tissue to Barrett's esophagus and the changes in the esophagus. We compared the relationship between these three (Normal tissue / Barretts esophagus / adenocarcinoma / ,156 / 28 / 41). The results showed that the CPD gene was significantly more highly expressed in esophageal adenocarcinoma tissue than in normal tissue ( Figure 4 A). At the same time, the ROC curve shows a significant discrimination effect ( Figure 4 B). There was no significant difference in CPD between Barrett's esophagus and adenocarcinoma ( Figure 4 C) and discrimination effect ( Figure 4 D). In contrast, Barrett's esophagus showed high expression of CPD compared with normal tissues ( Figure 4 E), and the diagnostic ROC curve showed a critical diagnostic performance ( Figure 4 F) This indicates the possible cancerous potential of Barrett's esophagus.

[0104] (4) Analysis of CPD gene expression in esophageal adenocarcinoma and esophageal squamous cell carcinoma

[0105] Due to the small number of esophageal adenocarcinoma cases, cross-validation cannot be performed. This example focuses on the overall data set to explore expression analysis, esophageal adenocarcinoma (AD) and esophageal squamous cell carcinoma (SQC) (adenocarcinoma / squamous cell carcinoma, 41 / 116). The results showed that the expression level of CPD gene in esophageal adenocarcinoma tissue was lower than that in esophageal squamous cell carcinoma ( Figure 5 A). At the same time, the ROC curve shows a significant discrimination effect ( Figure 5 B). This suggests that CPD has the function of differential diagnosis between esophageal squamous cell carcinoma and adenocarcinoma.

[0106] (5) Biological function analysis of CPD gene in esophageal adenocarcinoma and esophageal squamous cell carcinoma

[0107] This example analyzes the biological significance of CPD in tumor tissue in esophageal adenocarcinoma and esophageal squamous cell carcinoma. We used Pearson correlation to analyze the associated genes of CPD and performed GSEA analysis by sorting the correlation coefficients. We found that in the Hallmark data, CPD activation in esophageal adenocarcinoma was associated with positive activation of important cancer features, such as proliferation KRAS, AKT malignant signaling pathways, and glycolytic phenotypes ( Figure 6 A), in esophageal squamous cell carcinoma, CPD showed positive correlation with the activation of malignant proliferation pathway, E2F malignant signaling pathway, AKT signaling pathway, KRAS signaling pathway and epithelial-mesenchymal transition ( Figure 6 B).

[0108] (6) CPD in esophageal cancer shows high protein expression

[0109] In 7 esophageal cancer tissue specimens in our department, immunohistochemistry revealed that CPD showed high expression of protein in cancer compared with adjacent adjacent tissues ( Figure 7 AG). We also used WB to detect the protein expression of CPD in four pairs of esophageal cancer-adjacent tissues. Similarly, CPD showed obvious high protein expression in cancer ( Figure 7 H).

[0110] Example 2 Analysis of the diagnostic efficacy of CPD in real clinical samples collected by the present invention

[0111] 1. Sample source

[0112] In addition, 40 esophageal cancer tissue samples and 40 corresponding paracancerous tissue samples were collected in our department (Department of Thoracic Surgery, The Second Affiliated Hospital of Air Force Medical University) for further verification, esophageal cancer tissue: paracancerous tissue = 40 cases: 40 cases.

[0113] 2. Experimental methods

[0114] In this example, the tissue chip method was used to further verify the expression of CPD in the above-mentioned real clinical sample set, and the area under the receiver operating characteristic curve (AUC) was calculated through ROC analysis to analyze the accuracy (AUC value), sensitivity and specificity of CPD in diagnosing esophageal cancer, so as to further determine the diagnostic efficacy of CPD in the above-mentioned real clinical sample set.

[0115] Tissue chip method: Tissue microarray method, multiple different individual tissue specimens are arranged in a regular array on the same slide for in situ histological study with unified indicators. 40 cases of esophageal cancer tissues and adjacent tissues were taken, fixed with 4% paraformaldehyde, dehydrated with gradient ethanol, transparentized with xylene, immersed in wax, and embedded in paraffin. After the paraffin sections were dried, dewaxed with xylene, and hydrated with gradient ethanol, they were washed with phosphate buffer and microwave heated for antigen repair. After 3% hydrogen peroxide treatment of endogenous peroxidase, after blocking, the primary antibody was incubated overnight (anti-CPD antibody: DF9339, Affinity Biosciences, OH, USA), washed the next day, incubated with enzyme-labeled secondary antibody, washed, and DAB was used for color development. The protein content in immunohistochemistry was measured by semi-quantitative histochemical method: H-score = Σ(pi*i), "pi" represents the percentage of positive cells, and "I" represents the signal intensity.

[0116] 3. Experimental results

[0117] The results of tissue microarray analysis were as follows Figure 8 As shown in Figure 2, tissue microarray analysis revealed that CPD protein was significantly more expressed in esophageal cancer tissues than in adjacent adjacent tissues. The results of differential expression analysis and diagnostic efficacy validation are shown in Figure 2. Fig. 9 As shown in the results, the expression level of CPD in esophageal cancer tissue was significantly higher than that in adjacent cancer tissue. At the same time, the ROC curve analysis results showed a significant differentiation effect, indicating that CPD can be used for the effective diagnosis of esophageal cancer with high accuracy, with an AUC value of 0.9319, a specificity of 90%, and a sensitivity of 85%.

Claims

1. Application of a reagent for detecting the protein expression level of the biomarker CPD in a sample in the preparation of a product for diagnosing esophageal cancer; The reagent is an antibody that specifically binds to the protein encoded by the CPD.

2. The use according to claim 1, characterized in that: The sample is a tissue sample derived from a subject.

3. Use of a reagent for detecting the protein expression level of the biomarker CPD in a sample in the preparation of a product for diagnosing esophageal squamous cell carcinoma; The reagent is an antibody that specifically binds to the protein encoded by the CPD.

4. The use according to claim 3, characterized in that: The sample is a tissue sample derived from a subject.

5. Use of a reagent for detecting the protein expression level of CPD in a sample in the preparation of a system or device for diagnosing esophageal cancer; The reagent is an antibody that specifically binds to the protein encoded by the CPD.

6. Use of a reagent for detecting the protein expression level of CPD in a sample in the preparation of a system or device for diagnosing esophageal squamous cell carcinoma; The reagent is an antibody that specifically binds to the protein encoded by the CPD.

7. A system for diagnosing esophageal cancer, characterized in that: The system includes a processor, an input module, and an output module; Among them, the input module is used to input the protein expression level of CPD in the subject's sample; the processor is used to perform logical operations on the input information using bioinformatics methods, and the processor contains a computer-readable medium storing instructions, and the instructions execute an algorithm on the input CPD protein expression level when executed by the processor; the output module is used to output the results of whether the subject has esophageal cancer and the risk of esophageal cancer.

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