Application of CREPT as a marker for predicting therapeutic response to anti-gastric cancer immunotherapy

Abstract

The present invention relates to the application of CREPT gene expression as a therapeutic response indicator for predicting anti-gastric cancer immunotherapy. The expression level of CREPT predicts the therapeutic response effect of gastric cancer immunotherapy. When the expression of CREPT gene and PD-L1 are further used as therapeutic response indicators for predicting anti-gastric cancer immunotherapy, low CREPT expression level and PD-L1 expression predict good therapeutic response effect of gastric cancer immunotherapy. The therapeutic response indicator of the present invention has high sensitivity and specificity and can accurately predict the therapeutic response effect of anti-gastric cancer immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of CREPT as an indicator for predicting the therapeutic response of anti-gastric cancer immunotherapy, and the application of a reagent for detecting CREPT expression in the preparation of a kit for predicting the response of gastric cancer immunotherapy. Background Art

[0002] CREPT (Cell cycle-related and expression elevated protein in tumor) is a cancer-related gene. Compared with adjacent and normal tissues, it is highly expressed in a variety of tumor tissues including liver cancer, gastric cancer, pancreatic cancer, glioma, prostate cancer and colorectal cancer, and is negatively correlated with patient prognosis (Lu et al., 2012). Many research groups have reported consistent phenomena in different tumors, that is, overexpression of CREPT promotes tumor growth, while inhibition or knockout of CREPT has the opposite effect (Li et al., 2021; Lu et al., 2012). Moreover, CREPT plays an important role in transcriptional regulation. It can not only interact with RNA polymerase II (RNAP II) to regulate the expression of target genes such as cyclinD1, but also participate in the regulation of multiple signaling pathways such as Wnt and STAT3, thereby promoting cell growth and tumorigenesis (Lu et al., 2012). Many signs indicate that CREPT is a promising new target for the treatment of tumors.

[0003] However, there is currently no reliable treatment response indicator for the immunotherapy of gastric cancer, and it is also unknown how CREPT participates in the immunotherapy of gastric cancer. Therefore, it is necessary to study the role of CREPT in the immunotherapy of gastric cancer, so as to design targeted biological products to meet the clinical and scientific research needs of detecting the immunotherapy response of gastric cancer. Summary of the invention

[0004] In response to the problems existing in the above-mentioned prior art, the present application provides the application of CREPT gene expression as an indicator for predicting the therapeutic response of anti-gastric cancer immunotherapy, wherein the expression level of CREPT predicts the therapeutic response effect of gastric cancer immunotherapy, a high expression level of CREPT predicts a poor therapeutic response effect of gastric cancer immunotherapy, and a low expression level of CREPT predicts a good therapeutic response effect of gastric cancer immunotherapy.

[0005] CREPT, also known as RPRD1B or C20ORF77, is a gene found in recent years and located on human chromosome 20. In humans, the CREPT gene contains 5 exons and encodes a protein composed of 312 amino acids. CREPT is expressed at high levels in many tumors, promoting cell proliferation and tumor formation. CREPT has a promoting effect on a variety of malignant tumors such as colorectal cancer, pancreatic cancer, and gastric cancer. It can participate in the regulation of multiple signaling pathways, such as Wnt, MAPK, STAT3, and NF-κB.

[0006] Gastric cancer is a malignant tumor originating from the gastric mucosal epithelium. Gastric cancer can occur in any part of the stomach, and more than half of them occur in the gastric antrum. The greater and lesser curvatures of the stomach and the anterior and posterior walls can be affected. The vast majority of gastric cancers are adenocarcinomas. There are no obvious symptoms in the early stage, or non-specific symptoms such as upper abdominal discomfort and belching may occur. They are often similar to the symptoms of chronic gastric diseases such as gastritis and gastric ulcers, and are easily overlooked. It can be divided into adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, carcinoid, etc., and the vast majority are gastric adenocarcinomas. According to different tissue structures, adenocarcinomas can also be divided into papillary carcinoma, tubular adenocarcinoma, poorly differentiated adenocarcinoma, mucinous adenocarcinoma and signet ring cell carcinoma.

[0007] The therapeutic response to immunotherapy is characterized by a good immunotherapy response or a good prognosis for individuals with cancers that are sensitive to immunotherapy (e.g., immune checkpoint blockade therapy), and a poor immunotherapy response or a poor prognosis for individuals with cancers that are resistant to immunotherapy (e.g., immune checkpoint blockade therapy). Predicting a patient's response to immunotherapy is intended to assess the likelihood that a patient will experience a positive or negative outcome to a particular immunotherapy.

[0008] As certain embodiments of the present application, the expression of the CREPT gene is used together with the expression of PD-L1 as an indicator for predicting the therapeutic response of gastric cancer immunotherapy, and the low expression level of CREPT and the expression of PD-L1 predict a good therapeutic response effect of gastric cancer immunotherapy.

[0009] The present invention also provides the use of a detection reagent in preparing a kit for predicting gastric cancer immunotherapy response, wherein the detection reagent comprises: (1) a reagent capable of detecting CREPT expression.

[0010] As certain embodiments of the present application, the detection reagent further includes (2) a reagent capable of detecting PD-L1 expression.

[0011] As certain embodiments of the present application, the reagent capable of detecting the expression of CREPT is a reagent capable of detecting CREPT protein or CREPT mRNA. As certain embodiments of the present application, the reagent capable of detecting the expression of PD-L1 is a reagent capable of detecting PD-L1 protein or PD-L1 mRNA.

[0012] Determining the level of gene expression is to determine the expression level of the expression product of the gene, and the expression level can be determined at the nucleic acid level or the protein level. The expression level of the relevant gene in the individual to be predicted is compared with the expression level of the relevant gene in the comparable individual or sample to predict the therapeutic response effect of the individual immunotherapy to be predicted. The determination of the gene expression level can be the presence or amount of mRNA in the cancer cell sample determined. The method for doing so is well known to the technician, and the gene expression level can be any conventional method, for example, determined in the cancer cell sample using a nucleic acid microarray or nucleic acid synthesis (quantitative PCR).

[0013] In addition, the determination of gene expression levels can involve determining the level of protein expressed from the gene in a sample containing cancer cells derived from the individual to be predicted. Protein expression levels can be determined by any available technical means (including immunoassays). For example, expression levels can be determined by immunohistochemistry (IHC), Western blot, ELISA, immunoelectrophoresis, immunoprecipitation, flow cytometry, mass cytometry and immunostaining.

[0014] As certain embodiments of the present application, the reagent capable of detecting the expression of CREPT is a reagent capable of detecting the presence or expression level of CREPT protein.

[0015] As certain embodiments of the present application, the reagent capable of detecting the expression of CREPT is used to determine the presence or expression level of CREPT protein by in vitro detection.

[0016] As certain embodiments of the present application, the in vitro detection method is selected from immunological detection, mass spectrometry, and spectroscopy.

[0017] The basic principle of immunological detection is that cytokines (or receptors) are combined with corresponding specific antibodies (monoclonal antibodies or polyclonal antibodies), which are amplified and displayed through labeling techniques such as isotopes, fluorescence or enzymes, thereby qualitatively or quantitatively displaying the level of cytokines (or receptors). Immunological detection methods mostly use ELISA and RIA methods, which can be divided into the center method, competitive method and indirect method.

[0018] Mass spectrometry (MS) is a method of using electric and magnetic fields to separate moving ions (charged atoms, molecules or molecular fragments, including molecular ions, isotopic ions, fragment ions, rearranged ions, multi-charged ions, metastable ions, negative ions and ions generated by ion-molecule interactions) according to their mass-to-charge ratios for detection. Mass spectrometry can be used for qualitative analysis. For example, in pharmacological and biological research, the presence of drugs and metabolites can be determined based on their retention time and corresponding mass fragmentation diagrams on gas chromatograms. It can also be used for quantitative analysis, using the stable isotope isomers of the tested compound as internal standards to obtain more accurate results.

[0019] Spectroscopy is a type of instrumental analysis method that determines the properties, content and structure of a substance based on the electromagnetic radiation emitted by the substance or the radiation signal or signal change generated after the interaction between the substance and the radiation. According to the different ways of generating spectra, spectroscopy can be divided into absorption spectroscopy and emission spectroscopy. Absorption spectroscopy is a qualitative, quantitative and material structure analysis based on the absorption spectrum of the substance. The absorption spectrum is the spectrum generated by the absorption of the corresponding radiation energy by the substance. The necessary condition for its generation is that the energy provided just meets the energy required for the transition between the two energy levels of the absorbing substance. Absorption spectroscopy specifically includes Mossbauer spectroscopy, x-ray absorption spectroscopy, atomic absorption spectroscopy, ultraviolet-visible absorption spectroscopy, infrared absorption spectroscopy, and NMR. Emission spectroscopy is a qualitative, quantitative and material structure analysis using the absorption spectrum of the substance. Emission spectrum refers to the spectrum generated by the atoms, ions or molecules that constitute the substance being excited by radiation energy, thermal energy, electrical energy or chemical energy and transitioning to the excited state, and then releasing energy in the form of radiation when returning to the ground state from the excited state. Emission spectroscopy specifically includes: atomic emission spectroscopy, atomic fluorescence spectroscopy, molecular fluorescence spectroscopy and phosphorescence spectroscopy.

[0020] As certain embodiments of the present application, the immunological detection is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA), and radioimmunoassay (RIA).

[0021] Enzyme-Linked ImmunoSorbent Assay (ELISA) is a solid-phase immunoassay method for detecting trace substances in body fluids. ELISA has become a cutting-edge topic in the field of analytical chemistry. It is a special reagent analysis method and a new type of immunoassay technology developed on the basis of immunoenzymatic techniques. The specific operation is: to make the antigen or antibody bind to the surface of a certain solid phase carrier and maintain its immune activity; to make the antigen or antibody connect with a certain enzyme to form an enzyme-labeled antigen or antibody, which retains both its immune activity and enzyme activity. During the determination, the specimen to be tested (the antibody or antigen to be measured) and the enzyme-labeled antigen or antibody react with the antigen or antibody on the surface of the solid phase carrier in different steps. The antigen-antibody complex formed on the solid phase carrier is separated from other substances by washing, and finally the amount of enzyme bound to the solid phase carrier is in a certain proportion to the amount of the substance to be tested in the specimen. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme to become a colored product. The amount of the product is directly related to the amount of the substance being tested in the specimen, so qualitative or quantitative analysis can be performed based on the depth of the color reaction. Since the enzyme has a high catalytic frequency, the reaction effect can be greatly amplified, thereby achieving a high sensitivity of the determination method.

[0022] Electrochemical immunoassay (ECIA) uses the specific reaction of antigens and antibodies for detection. It is based on the principle of immunoassay and combines electrochemical detection technology with immune reaction to achieve quantitative analysis of specific substances. Electrochemical immunoassay involves labeling some substances that are easy to measure and highly sensitive onto specific antigens or antibody molecules, and using the enhanced amplification effect of these markers to display the properties and content of the antigens or antibodies in the reaction system. This method has high sensitivity and high specificity, and can perform quantitative analysis without the use of radioactive substances. Although it has poor stability and reproducibility, it has a wide range of applications in clinical diagnosis and other fields due to its high sensitivity and the advantages of no radioactive substances.

[0023] Chemiluminescence immunoassay (CLIA) is a technique that combines highly sensitive chemiluminescence measurement technology with highly specific immune reactions, and is used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs. Chemiluminescence immunoassay consists of two parts, namely the immune reaction system and the chemiluminescence analysis system. The chemiluminescence analysis system uses the catalysis of the chemiluminescent substance and the oxidation of the oxidant to form an excited intermediate. When this excited intermediate returns to a stable ground state, it simultaneously emits a photon (hM), and the light quantum yield is measured using a luminescence signal measuring instrument. The immune reaction system is to directly label the luminescent substance (an excited intermediate is generated under the excitation of the reactant) on the antigen (chemiluminescence immunoassay) or antibody (immunochemiluminescence assay), or the enzyme acts on the luminescent substrate.

[0024] Electrochemi-luminescence immunoassay is an immunoassay technique that uses terpyridine ruthenium to label antigens or antibodies and utilizes the principle of electrochemical chemiluminescence. The basic principle is that tripropylamine and terpyridine ruthenium in the immune reaction complex lose electrons around the electrode to form trivalent free radicals, and the excited terpyridine ruthenium emits photons with a wavelength of 620nm when de-excited, and repeatedly generates more photons on the electrode surface. The measurement of these photons can enhance the signal of the object being detected.

[0025] Fluorescence immunoassay (FIA) is a method that combines the specificity of immunological reactions with the sensitivity of fluorescence technology. Fluorescence immunoassay plays an important role in basic medical research and clinical diagnosis, and the development of labeled probes with excellent performance is the decisive factor in the development of this technology. Fluorescence immunoassay has the advantages of strong specificity, high sensitivity and good practicality, so it is used to measure low-content biologically active compounds, such as proteins (enzymes, receptors, antibodies), hormones (steroids, thyroid hormones, peptide hormones), drugs and microorganisms. There are two modes of fluorescence immunoassay, namely competitive type and sandwich type. Among them, the competitive type (taking the competitive type of labeled antigen as an example) is an immunoassay based on the competition between unlabeled antigen (Ag) and labeled antigen (Ag-L) for binding to limited antibodies (Ab). During the detection, the concentrations of Ab and Ag-L are fixed. When unlabeled Ag is added to the immune mixture of Ab and Ag-L, the binding of Ag and Ab reduces the amount of immune complexes of Ag-L and Ab. For sandwich immunoassay, the reaction principle is to fix an excess of Ab on the carrier of the immune reaction, then add a certain amount of Ag, and after the immune reaction, add an excess of labeled antibody (Ab-L) to form a "sandwich" sandwich immune complex. The more Ag there is in the sample, the more Ab-L is bound, and the stronger the labeled fluorescent signal of the sandwich immune complex.

[0026] As a type of biochip, protein chip has become an important tool for studying proteins. The detection principle of protein chip is the same as immunoassay, and it can also be called chip immunoassay. Protein chips are roughly divided into three types: chips composed of protein microarrays, microfluidic chips based on various microstructures, and suspension chips combining microsphere encoding and flow detection. Among these three types, the ones that use the immune principle and fluorescence detection are the mainstream. Therefore, this type of protein chip technology can be called fluorescent immunoassay on the chip. Due to its advantages of small sample size, high analysis throughput, and the ability to perform simultaneous analysis of multiple components, it has become an important development direction for fluorescent immunoassay and even the entire immunoassay.

[0027] Radioimmunoassay (RIA) is a radionuclide in vitro detection method that combines the measurement method of radionuclides with the basic principle of immune response. This method has the advantages of high sensitivity, strong specificity, good accuracy and small sample usage, so it has developed rapidly. This test technology is not only widely used to determine antigenic proteins, enzymes and polypeptide hormones, but also increasingly widely used to determine many drugs that are not antigenic themselves. In a certain volume, there is a certain amount of antibody and a certain amount of radioactive antigen. The more non-radioactive antigen is added, the less radioactive antigen is bound to the antibody. By separating the antibody bound to the antigen from the unbound antigen, and then measuring the radioactivity of these antibodies by measuring the radioactivity, the antigen can be quantitatively analyzed.

[0028] As certain embodiments of the present application, the reagent capable of detecting the expression of CREPT comprises an antibody capable of specifically binding to CREPT.

[0029] As certain embodiments of the present application, the reagent capable of detecting the expression of CREPT comprises an antibody capable of specifically binding to CREPT and a detectable label linked to the antibody.

[0030] As certain embodiments of the present application, the reagent capable of detecting the expression of CREPT comprises an antibody capable of specifically binding to CREPT, and a second antibody that specifically recognizes the antibody; optionally, the second antibody further comprises a detectable label.

[0031] As certain embodiments of the present application, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds), an electrochemiluminescent agent (e.g., terpyridine ruthenium), an electroactive substance, a fluorescent dye (e.g., FITC, TRITC, PE, Texas Red or a cyanine dye derivative (e.g., Cy7, Alexa 750)), a radionuclide (e.g., radioactive sulfone), and biotin.

[0032] As certain embodiments of the present application, the reagent capable of detecting the expression of CREPT is a reagent capable of detecting the presence or level of CREPT mRNA.

[0033] As certain embodiments of the present application, the reagent capable of detecting the expression of CREPT is used to determine the presence or level of CREPT mRNA by in vitro detection.

[0034] As certain embodiments of the present application, the in vitro detection method is selected from qPCR, nucleic acid sequencing or hybridization, mass spectrometry, and chromatography.

[0035] Quantitative polymerase chain reaction (qPCR) is a technique for selectively amplifying and quantitatively detecting DNA or complementary DNA (cDNA) regions. Oligonucleotide primers located on both sides of the region of interest are used to amplify the sequence using DNA polymerase. Fluorescent dyes or fluorescent groups are added to the PCR amplification reaction system. During the entire PCR process, the changes in the amount of amplified products in each cycle are monitored in real time by collecting fluorescent signals. Finally, the sample to be tested is quantitatively analyzed using the standard curve and CT value. There are two methods for quantitative polymerase chain reaction: SYBR Green method and TaqMan method.

[0036] Nucleic acid hybridization: also known as nucleic acid molecular hybridization technology, refers to the process in which complementary nucleotide sequences (DNA and DNA, DNA and RNA, RNA and RNA, etc.) form non-covalent bonds through Watson-Crick base pairing, thereby forming stable homologous or heterologous double-stranded molecules. Using the basic properties of DNA denaturation and renaturation, molecular hybridization technology is combined with blotting technology and probe technology in order to conduct qualitative or quantitative analysis of DNA / RNA.

[0037] Nucleic acid sequencing refers to the determination of the base arrangement order of nucleic acid to decipher the genetic code of nucleic acid, including the initial Sanger sequencing, the later NGS, and the current single-molecule sequencing technology.

[0038] Mass spectrometry refers to an analytical method that measures the mass-to-charge ratio of ions. It ionizes the components in the sample in the ion source to generate charged ions with different mass-to-charge ratios. After being accelerated by an electric field, an ion beam is formed and enters the mass analyzer. In the mass analyzer, the electric and magnetic fields are used to cause opposite velocity dispersion, and they are focused separately to obtain a mass spectrum, thereby determining their mass.

[0039] Chromatography, also known as chromatography or chromatographic method, is a physical and chemical analysis method that uses the difference in the forces (distribution, adsorption, ion exchange, etc.) between different solutes (samples) and the stationary phase and the mobile phase. When the two phases move relative to each other, each solute undergoes multiple equilibriums between the two phases, so that each solute is separated from each other. The purpose of analytical chromatography is to quantitatively or qualitatively determine the properties and content of each component in a mixture. Qualitative analytical chromatography includes thin layer chromatography, paper chromatography, etc., and quantitative analytical chromatography includes gas chromatography, high performance liquid chromatography, etc. The application of chromatography in the field of analysis combines the separation and determination processes into one, reduces the difficulty of mixture analysis, and shortens the analysis cycle. It is a more mainstream analysis method.

[0040] As certain embodiments of the present application, the reagent capable of detecting the expression of CREPT comprises a primer and / or a probe that specifically binds to CREPT mRNA.

[0041] As certain embodiments of the present application, the reagent capable of detecting the expression of PD-L1 is a reagent capable of detecting the presence or expression level of PD-L1 protein.

[0042] As certain embodiments of the present application, the reagent capable of detecting the expression of PD-L1 determines the presence or expression level of PD-L1 protein by in vitro detection.

[0043] As certain embodiments of the present application, the in vitro detection method is selected from immunological detection, mass spectrometry, and spectroscopy.

[0044] As certain embodiments of the present application, the immunological detection is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA), and radioimmunoassay (RIA).

[0045] As certain embodiments of the present application, the reagent capable of detecting the expression of PD-L1 comprises an antibody capable of specifically binding to PD-L1.

[0046] As certain embodiments of the present application, the reagent capable of detecting the expression of PD-L1 comprises an antibody capable of specifically binding to PD-L1 and a detectable label linked to the antibody.

[0047] As certain embodiments of the present application, the reagent capable of detecting the expression of PD-L1 comprises an antibody capable of specifically binding to PD-L1, and a second antibody that specifically recognizes the antibody. As certain embodiments of the present application, the second antibody also includes a detectable label.

[0048] As certain embodiments of the present application, the detectable label is selected from enzymes, chemiluminescent agents, electrochemiluminescent agents, electroactive substances, fluorescent dyes, radionuclides, and biotin.

[0049] As some embodiments of the present application, the enzyme includes horseradish peroxidase or alkaline phosphatase. As some embodiments of the present application, the chemiluminescent reagent includes acridinium ester compounds. As some embodiments of the present application, the electrochemiluminescent reagent includes terpyridine ruthenium. As some embodiments of the present application, the fluorescent dye includes FITC, TRITC, PE, Texas Red and cyanine dye derivatives. As some embodiments of the present application, the radionuclide includes radioactive sulfonate. As some embodiments of the present application, the cyanine dye derivative includes Cy3, Cy5, Cy7, and Alexa 750.

[0050] As certain embodiments of the present application, the reagent capable of detecting the expression of PD-L1 is a reagent capable of detecting the presence or level of PD-L1 mRNA.

[0051] As certain embodiments of the present application, the reagent capable of detecting the expression of PD-L1 determines the presence or level of PD-L1 mRNA by in vitro detection.

[0052] As certain embodiments of the present application, the in vitro detection method is selected from qPCR, nucleic acid sequencing or hybridization, mass spectrometry, and chromatography.

[0053] As certain embodiments of the present application, the reagent capable of detecting the expression of PD-L1 comprises a primer and / or a probe that specifically binds to PD-L1 mRNA.

[0054] As certain embodiments of the present application, the expression level of the CREPT is determined by Allred Score. As certain embodiments of the present application, the high expression level of the CREPT is determined by Allred Score score of 4-8 points. As certain embodiments of the present application, the low expression level of the CREPT is determined by ALL score score of 0-3 points.

[0055] The Allred Score is a scoring system for evaluating estrogen receptor (ER) expression that combines immunohistochemical staining results with pathological evaluation to predict the prognosis of diseases such as endometrial cancer.

[0056] In certain embodiments, the level of CREPT expression is determined by H-score. In certain embodiments, the level of CREPT expression is determined by Quick score.

[0057] As certain embodiments of the present application, the sample for detecting CREPT and / or PD-L1 is a tissue.

[0058] As certain embodiments of the present application, gastric cancer includes adenocarcinoma.

[0059] As certain embodiments of the present application, the sample with low CREPT expression level predicts a good response to gastric cancer immunotherapy in the patient from which the sample originated.

[0060] As certain embodiments of the present application, the sample with low CREPT expression level and positive PD-L1 predicts a good response to gastric cancer immunotherapy in the patient from which the sample originated.

[0061] The present application also provides a kit for predicting the response to immunotherapy of gastric cancer, the kit comprising: (1) a reagent capable of detecting the expression of CREPT; and (2) a reagent capable of detecting the expression of PD-L1.

[0062] As certain embodiments of the present application, the reagent capable of detecting the expression of CREPT is a reagent capable of detecting the presence or expression level of CREPT protein; the reagent capable of detecting the expression of CREPT is a reagent capable of detecting the presence or expression level of CREPT mRNA.

[0063] As certain embodiments of the present application, the reagent capable of detecting PD-L1 expression is a reagent capable of detecting PD-L1 protein or PD-L1 mRNA.

[0064] The present application also provides the application of CREPT gene expression in screening immunotherapy methods for gastric cancer immunotherapy, the expression level of the CREPT predicts the therapeutic response effect of the immunotherapy method for gastric cancer immunotherapy, a high expression level of CREPT predicts a poor immunotherapy method response effect for gastric cancer immunotherapy, and a low expression level of CREPT predicts a good immunotherapy method response effect for gastric cancer immunotherapy, the high expression level of CREPT is 4-8 points as determined by the AllredScore score, and the low expression level of CREPT is 0-3 points as determined by the ALL score score.

[0065] As certain embodiments of the present application, the expression of the CREPT gene is used together with the expression of PD-L1 in screening immunotherapy methods for gastric cancer immunotherapy, and the low expression level of CREPT and PD-L1 expression predict a good response effect of the immunotherapy method for gastric cancer immunotherapy.

[0066] As described above, the application of CREPT of the present application as an indicator for predicting the therapeutic response of anti-gastric cancer immunotherapy has the following beneficial effects:

[0067] (1) This application is the first to propose a marker that can accurately predict the therapeutic response index of anti-gastric cancer immunotherapy. Before this application, Ki67 had no indicative effect on gastric cancer immunotherapy;

[0068] (2) The CREPT of the present application, whether used alone as a marker, or the expression of the CREPT gene combined with the expression of PD-L1 as a therapeutic response indicator for gastric cancer immunotherapy, has high sensitivity and specificity, and meets the requirements of clinical testing and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The overall expression pattern of CREPT in gastric cancer is shown;

[0070] Figure 2 It showed that patients with low CREPT expression levels responded better to immunotherapy;

[0071] Figure 3 It shows that PD-L1 positive patients have good immunotherapy effects, but the proportion of positive patients is small;

[0072] Figure 4 It was shown that Ki67 has no indicative effect on immunotherapy;

[0073] Figure 5 The analysis of CREPT in combination with PD-L1 as an immunotherapy marker was shown;

[0074] Figure 6 Evaluation of CREPT and PD-L1 alone or in combination as immunotherapy markers is shown. DETAILED DESCRIPTION

[0075] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0076] [Experimental Materials]

[0077] Gastric cancer tissue chip: Zhongke Photochemical D160St01

[0078] Hematoxylin: Sigma 03971

[0079] PD-L1 antibody: abcam, ab205921 (dilution ratio 1:500)

[0080] Ki67 antibody: abcam, ab15580 (dilution ratio 1:5000)

[0081] CREPT antibody: 4H1 (developed by the applicant, dilution ratio 1:500)

[0082] HPR rabbit / mouse:Dako, 41654290

[0083] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0084] Embodiment 1:

[0085] Global expression pattern of CREPT in gastric cancer

[0086] By performing CREPT histochemical staining on gastric cancer tissue chips, the applicant found that CREPT was not expressed in normal gastric mucosa, and there were patients with different degrees of CREPT positive and negative in patients with gastric cancer in stage I, II, and III. The representative staining results are shown in the figure below. Figure 1 As shown in A. Figure 1 A shows IHC staining of normal gastric mucosal tissue and IHC staining of gastric mucosal tissue of patients at different stages of gastric cancer development.

[0087] Furthermore, the applicant performed Allred scoring on the staining results of 8 stage I patients, 16 stage II patients, and 54 stage III patients. The results are as follows: Figure 1 As shown in B.

[0088] Allred Score is a scoring system for evaluating estrogen receptor (ER) expression. It combines immunohistochemical staining results and pathological evaluation to predict the prognosis of diseases such as endometrial cancer. The calculation of Allred Score is based on the score of IHC staining intensity and the proportion of positive cells. The specific steps are as follows:

[0089] Staining intensity score: It is divided into four levels according to the staining intensity, namely, no positive staining (negative) is scored as 0 points, light yellow (weak positive) is scored as 1 point, brownish yellow (positive) is scored as 2 points, and brownish brown (strong positive) is scored as 3 points;

[0090] Positive cell ratio: evaluates the percentage of positive cells in all tumor cells, usually divided into the following levels: 0% (0 points), 1%-10% (1 point), 11%-50% (2 points), 51%-80% (3 points), >80% (4 points).

[0091] Comprehensive score: Add the staining intensity score and the positive cell ratio score to get a total score. For example, if the staining intensity score is 2 (moderate intensity) and the positive cell ratio is 3 (51%-80%), the Allred Score is 5.

[0092] The calculation of the Allred Score is intended to provide a quantitative indicator for comparing ER expression levels between different samples, thereby helping doctors assess the prognosis of patients.

[0093] Next, through further statistics, it was found that among patients with stage I gastric cancer, CREPT-negative patients accounted for about 37.5%, weak-positive patients accounted for about 25%, positive patients accounted for about 25%, and strongly positive patients accounted for 12.5%; among patients with stage II gastric cancer, CREPT-negative patients accounted for about 43.8%, weak-positive patients accounted for about 12.5%, positive patients accounted for about 31.2%, and strongly positive patients accounted for 12.5%; among patients with stage III gastric cancer, CREPT-negative patients accounted for about 22.2%, weak-positive patients accounted for about 27.9%, positive patients accounted for about 25.9%, and strongly positive patients accounted for 245% ( Figure 1 C).

[0094] According to the above experimental content, it can be seen that the expression intensity of CREPT in gastric cancer is divided into four levels, namely strong positive (+++), positive (++), weak positive (+), and negative (-); and CREPT is strongly positive, positive, weakly positive, and negative in patients with stage I, II, and III gastric cancer (see Figure 1 B-1C). The expression intensity of CREPT has no relationship with the development stage of gastric cancer. In addition, although CREPT is highly expressed in tumors, it is not positive in every patient. It is worth further exploring what indicators can be used to distinguish CREPT-positive and -negative patients in clinical practice.

[0095] Embodiment 2:

[0096] Patients with low CREPT expression levels respond better to immunotherapy

[0097] Pathological sections of 28 patients with gastric cancer who received immunotherapy were collected and subjected to CREPT immunohistochemical staining. Representative results are shown in Figure 2. Figure 2 As shown in A. It can be seen that there are differences in the expression of CREPT in patients with SD (Stable Disease), PR (Partial Response), and PD (Progression Disease).

[0098] Among the pathological sections of 28 patients receiving immunotherapy, there was 1 complete remission (CR), 18 partial remissions (PR), 4 stable disease (SD) and 5 progressive disease (PD) patients. The Allred score of immunohistochemical staining was further performed. The applicant found that CREPT was negative in patients with complete remission, partial remission and stable disease, while CREPT was strongly positive in patients with progressive disease, which provided strong support for the response effect of CREPT in the indication of immunotherapy. Furthermore, the applicant scored and counted the CREPT immunohistochemical results of all patients ( Figure 2B). The intensity of CREPT expression was higher in PD patients, while in PR patients, the intensity of CREPT expression was distributed in all levels, and the intensity of CREPT expression in SD patients was between the two.

[0099] The statistical results show that the CREPT scores are relatively low in patients with complete remission, partial remission and stable disease (the applicant collectively refers to these three levels of response as disease control patients (DC)), while the scores are higher in patients with progressive disease. Among patients with disease control (DC), the population with low CREPT expression accounts for about 70% ( Figure 2 C).

[0100] Subsequently, the ROC curve was drawn, with the X-axis representing specificity (false positive rate) and the Y-axis representing sensitivity (accuracy), and the area under the curve (AUC) was further calculated, with the AUC value reaching 0.86 ( Figure 2 D), indicating that CREPT has good predictive ability in distinguishing positive and negative samples, suggesting that CREPT has great potential as a biomarker for immunotherapy.

[0101] Embodiment 3:

[0102] PD-L1 staining in patients undergoing immunotherapy

[0103] Currently, the more mature immunotherapy marker in clinical practice is PD-L1. In order to further evaluate the potential of CREPT as an immunotherapy marker, PD-L1 immunohistochemical staining was also performed on pathological sections of 28 gastric cancer patients undergoing immunotherapy. Figure 3 A shows the PDL1 staining results of 2 PR patients (1 PD-L1 positive, 1 PD-L1 negative) and 2 PD patients.

[0104] The PD-L1 expression in 28 patients undergoing advanced immunotherapy was statistically analyzed. Patients with PD-L1 positive responses to immunotherapy were all good, as assessed by the CPS score, which is the total number of PD-L1 positive cells / total number of all tumor cells.

[0105] CPS score is the abbreviation of tumor immune comprehensive positive score, which is used to evaluate the effectiveness of PD-1 / PD-L1 monoclonal antibody immunotherapy. CPS score is one of the important indicators to determine whether PD-1 / PD-L1 monoclonal antibody drugs are effective in treating tumors. CPS score adopts a percentage system. The larger the value, the higher the sensitivity of the tumor to immunotherapy and the better the efficacy. CPS score is a commonly used method to evaluate the expression rate of PD-L1 positive cells in clinical practice. The calculation method is to add the number of PD-L1 positive tumor cells to the number of PD-L1 positive immune cells, divide it by the total number of tumor cells and multiply it by 100. According to this algorithm, if CPS ≥ 5, the effect of immunotherapy is better; if CPS < 5, the effect of immunotherapy is poor (reference: Analyses of PD-L1 and Inflammatory Gene Expression Association With Efficacy of Nivolumab+ / -Ipilimumab in Gastric Cancer / Gastroesophageal Junction Cancer.).

[0106] PD-L1 positive patients have a good response to immunotherapy, but the proportion of PD-L1 positive patients is small, only 26% ( Figure 3 B, 3C); PD-L1 negative patients can also benefit from immunotherapy. In patients who do not respond well to immunotherapy, PD-L1 expression is negative ( Figure 3 B, 3C).

[0107] Example 4

[0108] 4.1Ki67 has no indication for immunotherapy

[0109] To further evaluate the potential of CREPT as an immunotherapy marker, Ki67 immunohistochemical staining was also performed on pathological sections of 28 gastric cancer immunotherapy patients. Representative results are shown in Figure 4 As shown in A. Figure 4 A shows the Ki67 tissue staining of gastric mucosal tissues in the PR and PD stages of two patients. The Ki67 expression of 28 patients with advanced gastric cancer who received immunotherapy was statistically analyzed and scored using Allred. The results showed that Ki67 was positive in both patients who responded well to immunotherapy and those who did not respond well to it ( Figure 4 B), no indication for immunotherapy.

[0110] 4.2 PD-L1 combined with CREPT for immunotherapy efficacy

[0111] Since Ki67 has no indication for immunotherapy, the efficacy of immunotherapy using PD-L1 in combination with CREPT was also verified.

[0112] The above experimental results can be found in Figure 5 , CREPT and PD-L1 are used together as immunotherapy markers to indicate the response to immunotherapy. Approximately 78% of responding patients are CREPT negative and PD-L1 positive (see Figure 5 A), compared with CREPT single marker, it is improved by 8%, and the AUC value of its ROC curve is 0.89 (see Figure 5 B).

[0113] 4.3 Comparison of CREPT and PD-L1 and their combination

[0114] To compare CREPT as a single marker and the combination of CREPT and PD-L1 as indicators for predicting treatment response to anti-gastric cancer immunotherapy, the applicant evaluated the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value using the following calculation formulas:

[0115] Sensitivity = true positive population / all disease control patients;

[0116] Specificity = true negative population / all patients with disease progression;

[0117] Accuracy = sum of true positives and true negatives / all patients;

[0118] Positive predictive value = true positive population / test positive population;

[0119] Negative predictive value = true negative population / test negative population;

[0120] The results are shown in Table 1 and Figure 6 .

[0121] Table 1 Comparison of CREPT and PD-L1 and their combination as immunotherapy markers

[0122] CREPT PD-L1 CREPT / PD-L1 Combination Sensitivity 70％ 26％ 78％ Specificity 100％ 100％ 100％ Accuracy 75％ 39％ 82％ Positive predictive value 100％ 100％ 100％ Negative predictive value 42％ 23％ 50％

[0123] According to Table 1 and Figure 6 It can be seen that CREPT single marker, as well as the combination of CREPT and PD-L1 as indicators for predicting treatment response to anti-gastric cancer immunotherapy, can reflect the detection results with high sensitivity and specificity. When CREPT and PD-L1 are used in combination as indicators, the detection sensitivity, accuracy, and negative predictive value are further improved.

[0124] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Application of CREPT gene expression as an indicator for predicting therapeutic response to anti-gastric cancer immunotherapy, characterized in that: The expression level of CREPT predicts the therapeutic response effect of gastric cancer immunotherapy. A high expression level of CREPT predicts a poor therapeutic response effect of gastric cancer immunotherapy, and a low expression level of CREPT predicts a good therapeutic response effect of gastric cancer immunotherapy. Preferably, the expression of the CREPT gene and the expression of PD-L1 are used together as indicators for predicting the therapeutic response of anti-gastric cancer immunotherapy, and a low expression level of the CREPT and PD-L1 expression predict a good therapeutic response effect of gastric cancer immunotherapy.

2. Use of the detection reagent in the preparation of a kit for predicting the response to gastric cancer immunotherapy, characterized in that: The detection reagent comprises: (1) Reagents capable of detecting CREPT expression; Preferably, the detection reagent further comprises (2) a reagent capable of detecting PD-L1 expression.

3. The use according to any one of claims 1 to 2, characterized in that: The reagent capable of detecting the expression of CREPT is a reagent capable of detecting CREPT protein or CREPT mRNA; The reagent capable of detecting PD-L1 expression is a reagent capable of detecting PD-L1 protein or PD-L1 mRNA.

4. The use according to claim 3, characterized in that: The reagent capable of detecting the expression of CREPT is a reagent capable of detecting the presence or expression level of CREPT protein; Preferably, the reagent capable of detecting the expression of CREPT determines the presence or expression level of CREPT protein by in vitro detection; Preferably, the in vitro detection method is selected from immunological detection, mass spectrometry, and spectroscopy; Preferably, the immunological test is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA), and radioimmunoassay (RIA); Preferably, the reagent capable of detecting the expression of CREPT comprises an antibody capable of specifically binding to CREPT; Preferably, the reagent capable of detecting the expression of CREPT comprises an antibody capable of specifically binding to CREPT and a detectable label linked to the antibody; Preferably, the reagent capable of detecting the expression of CREPT comprises an antibody capable of specifically binding to CREPT, and a second antibody that specifically recognizes the antibody; optionally, the second antibody further comprises a detectable label; Preferably, the detectable label is selected from an enzyme, a chemiluminescent agent, an electrochemiluminescent agent, an electroactive substance, a fluorescent dye, a radionuclide, and biotin; More preferably, the enzyme comprises horseradish peroxidase or alkaline phosphatase, the chemiluminescent reagent comprises acridinium ester compounds, the electrochemiluminescent reagent comprises terpyridine ruthenium, the fluorescent dye comprises FITC, TRITC, PE, Texas Red and cyanine dye derivatives, the radionuclide comprises radioactive sulfonate, and the cyanine dye derivatives comprise Cy3, Cy5, Cy7, and Alexa 750.

5. The use according to claim 3, characterized in that: The reagent capable of detecting the expression of CREPT is a reagent capable of detecting the presence or level of CREPT mRNA; Preferably, the reagent capable of detecting the expression of CREPT is used to determine the presence or level of CREPT mRNA by in vitro detection; Preferably, the in vitro detection method is selected from qPCR, nucleic acid sequencing or hybridization, mass spectrometry, chromatography; Preferably, the reagent capable of detecting the expression of CREPT comprises a primer and / or a probe that specifically binds to CREPT mRNA.

6. The use according to claim 3, characterized in that: The reagent capable of detecting the expression of PD-L1 is a reagent capable of detecting the presence or expression level of PD-L1 protein; Preferably, the reagent capable of detecting the expression of PD-L1 determines the presence or expression level of PD-L1 protein by in vitro detection; Preferably, the in vitro detection method is selected from immunological detection, mass spectrometry, and spectroscopy; Preferably, the immunological test is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent immunoassay (FIA), and radioimmunoassay (RIA); Preferably, the reagent capable of detecting the expression of PD-L1 comprises an antibody capable of specifically binding to PD-L1; Preferably, the reagent capable of detecting the expression of PD-L1 comprises an antibody capable of specifically binding to PD-L1 and a detectable label linked to the antibody; Preferably, the reagent capable of detecting the expression of PD-L1 comprises an antibody capable of specifically binding to PD-L1, and a second antibody that specifically recognizes the antibody; optionally, the second antibody further comprises a detectable label; Preferably, the detectable label is selected from an enzyme, a chemiluminescent agent, an electrochemiluminescent agent, an electroactive substance, a fluorescent dye, a radionuclide, and biotin; More preferably, the enzyme comprises horseradish peroxidase or alkaline phosphatase, the chemiluminescent reagent comprises acridinium ester compounds, the electrochemiluminescent reagent comprises terpyridine ruthenium, the fluorescent dye comprises FITC, TRITC, PE, Texas Red and cyanine dye derivatives, the radionuclide comprises radioactive sulfonate, and the cyanine dye derivatives comprise Cy3, Cy5, Cy7, and Alexa 750.

7. The use according to claim 3, characterized in that: The reagent capable of detecting the expression of PD-L1 is a reagent capable of detecting the presence or level of PD-L1 mRNA; Preferably, the reagent capable of detecting the expression of PD-L1 determines the presence or level of PD-L1 mRNA by in vitro detection; Preferably, the in vitro detection method is selected from qPCR, nucleic acid sequencing or hybridization, mass spectrometry, chromatography; Preferably, the reagent capable of detecting the expression of PD-L1 comprises a primer and / or a probe that specifically binds to PD-L1 mRNA.

8. The use according to claim 1, characterized in that: The expression level of the CREPT is determined by AllredScore; Or, the sample for testing CREPT and / or PD-L1 is tissue; or, gastric cancer including gastric adenocarcinoma; Preferably, the high expression level of CREPT is 4-8 points as determined by the Allred Score, and the low expression level of CREPT is 0-3 points as determined by the ALL score; Preferably, the sample with low CREPT expression level predicts a good gastric cancer immunotherapy response in the patient from which the sample originated; More preferably, the sample with low CREPT expression level and positive PD-L1 predicts a good response to gastric cancer immunotherapy in the patient from which the sample is derived.

9. A kit for predicting the response to immunotherapy of gastric cancer, characterized in that: The kit comprises: (1) a reagent capable of detecting CREPT expression; and (2) Reagents capable of detecting PD-L1 expression; Preferably, the reagent capable of detecting the expression of CREPT is a reagent capable of detecting the presence or expression level of CREPT protein; the reagent capable of detecting the expression of CREPT is a reagent capable of detecting the presence or expression level of CREPT mRNA; Preferably, the reagent capable of detecting PD-L1 expression is a reagent capable of detecting PD-L1 protein or PD-L1 mRNA.

10. Application of CREPT gene expression in screening immunotherapy methods for gastric cancer immunotherapy, characterized in that: The expression level of the CREPT predicts the therapeutic response effect of the immunotherapy method for gastric cancer immunotherapy. A high expression level of CREPT predicts a poor response effect of the immunotherapy method for gastric cancer immunotherapy, and a low expression level of CREPT predicts a good response effect of the immunotherapy method for gastric cancer immunotherapy. The high expression level of the CREPT is 4-8 points as determined by the Allred Score, and the low expression level of the CREPT is 0-3 points as determined by the ALL score. Preferably, the expression of the CREPT gene is used together with the expression of PD-L1 in screening immunotherapy methods for gastric cancer immunotherapy, and a low expression level of the CREPT and PD-L1 expression predict a good response effect of the immunotherapy method for gastric cancer immunotherapy.

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