Application of a reagent for detecting protein marker levels in the preparation of a diagnostic product for evaluating the effect of tumor treatment

Abstract

The present invention discloses the use of a reagent for detecting the level of a protein marker in the preparation of a diagnostic product for evaluating the effect of tumor treatment, and relates to the technical field of tumor treatment detection. PPIB protein can be used to monitor the effect of tumor treatment and to prognose recurrence. Compared with existing protein markers, PPIB protein has higher sensitivity and specificity for patient treatment monitoring and prognosis. By detecting or monitoring the level of PPIB protein in patients, non-invasive tumor diagnosis can be achieved, which is of great significance for tumor treatment monitoring and recurrence prognosis, improving the effectiveness of treatment decisions, and prolonging patient survival time.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment detection technology, and in particular to the use of a reagent for detecting protein marker levels in the preparation of a diagnostic product for evaluating tumor treatment effects. Background Art

[0002] Lung cancer is a major disease that seriously endangers human health. Among all malignant tumors, lung cancer has the highest mortality rate. Most patients are often diagnosed in the advanced stage and have missed the best time for surgical treatment. In the medical treatment of tumors, most patients can only receive chemotherapy, except for those with target mutations that can be targeted by targeted drugs and can be treated with targeted therapy, or those who meet the indications for immunotherapy and can be treated with immunotherapy. In addition, patients who fail first- and second-line targeted and immunotherapy must also undergo chemotherapy in subsequent treatment. Tumor treatment monitoring and recurrence prognosis are of great significance to improving the effectiveness of treatment decisions and prolonging patient survival.

[0003] Serum marker detection is a non-invasive tumor diagnosis method with great application prospects. Currently, serum markers widely used in clinical practice are mainly used for early diagnosis of tumors, and their sensitivity and specificity for patient treatment monitoring and prognosis are not high.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a reagent for detecting protein marker levels for use in the preparation of a diagnostic product for evaluating the efficacy of tumor treatment, thereby effectively monitoring the progress of tumor treatment and the prognosis of recurrence.

[0006] The present invention is achieved in that:

[0007] The present invention provides an application of a reagent for detecting the level of a protein marker in preparing a detection product or a diagnostic product for evaluating the therapeutic effect of a tumor, wherein the protein marker is PPIB.

[0008] Cyclophillin B (PPIB, also known as CypB) belongs to the cyclophilin family and is the intracellular receptor for the immunosuppressant cyclosporine A (CsA). It plays an important role in cells through the activity of peptidylprolyl cis-trans isomerase (PPIase) and molecular chaperones.

[0009] The inventors discovered that PPIB protein can be used as a protein marker for evaluating the effect of tumor treatment, and can be used for monitoring the effect of tumor treatment and prognosis of recurrence.

[0010] In a preferred embodiment of the present invention, the above-mentioned tumor is selected from malignant tumors.

[0011] In a preferred embodiment of the present invention, the above-mentioned tumor is selected from colorectal cancer, lung cancer, papilloma, gastric cancer, uterine cancer, ovarian cancer, cervical cancer, adenocarcinoma, breast cancer, adenoma, myeloma, glioma or squamous cell carcinoma.

[0012] In an alternative embodiment, the adenocarcinoma includes but is not limited to lung adenocarcinoma, thyroid cancer, salivary gland cancer, or pancreatic cancer.

[0013] In an alternative embodiment, the adenoma includes but is not limited to a cystadenoma, a fibroadenoma, a pleomorphic adenoma or a polypoid adenoma.

[0014] In an alternative embodiment, breast cancer includes but is not limited to breast ductal carcinoma, breast epithelial carcinoma or breast lobular carcinoma. Breast cancer, preferably stage II to IV and / or poorly differentiated invasive ductal carcinoma, comedo carcinoma and medullary carcinoma (preferably grade 2).

[0015] Ovarian cancer, serous and mucinous carcinomas (preferably stages Ic to IIIb), granulosa cell tumors, surface epithelial-stromal tumors (adenocarcinomas), cystadenocarcinomas, and endometrioid tumors.

[0016] Uterine cancer, preferably including endometrioid adenocarcinoma (preferably stage I to IIIc).

[0017] Bladder cancer, preferably including transitional cell carcinoma (preferably stage II to IV).

[0018] Lung cancer, preferably includes small cell lung cancer (preferably stage I to IIIb), non-small cell lung cancer (preferably poorly to moderately differentiated squamous and adenocarcinoma) and large cell lung cancer.

[0019] The squamous cell carcinoma is one or more of oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal cancer, esophageal squamous cell carcinoma (eg esophageal squamous cell carcinoma), lip squamous cell carcinoma, uterine squamous cell carcinoma, vaginal squamous cell carcinoma and skin squamous cell carcinoma.

[0020] Oral squamous cell carcinoma (OSCC) is also called oral squamous cell carcinoma. Examples of oral squamous cell carcinoma include but are not limited to tongue squamous cell carcinoma.

[0021] In other embodiments, the squamous cell carcinoma may also be squamous cell carcinoma formed by squamous metaplasia in the bronchus, bladder, renal pelvis and other parts.

[0022] In other embodiments, the above tumors include but are not limited to diseases induced by overexpression of PPIB protein.

[0023] PPIB protein markers, especially for monitoring treatment effects and assessing prognosis and recurrence of lung cancer, have high sensitivity and specificity.

[0024] In a preferred embodiment of the present invention, the above-mentioned tumor treatment is chemotherapy, immunotherapy, surgery, radiotherapy, anti-inflammatory therapy, targeted therapy and a combination thereof.

[0025] Immunotherapy, for example, targets immune checkpoint antigens, which preferably include PD-1, PD-L1, Tim3, LAG3, and CD47. Targets of targeted therapy preferably include growth factor receptors (EGFR) on the surface of tumor cells, intracellular signal transduction, and anti-tumor angiogenesis; targets of targeted therapy are, for example, selected from SIRPα.

[0026] The anti-inflammatory treatment comprises administering to the subject a cyclooxygenase inhibitor, optionally an inhibitor specific for cyclooxygenase-2.

[0027] In a preferred embodiment of the present invention, the above-mentioned product is selected from a reagent, a kit or a chip.

[0028] The chip is, for example, selected from a liquid phase chip. The application provided by the present invention can also be detected by a flow cytometer.

[0029] In a preferred embodiment of the present invention, when the above product is a kit, the kit includes a microplate, and the reagents for detecting the level of protein markers include PPIB protein standards, anti-PPIB protein antibodies and enzyme-labeled secondary antibodies.

[0030] In other embodiments, the detection method of the kit includes but is not limited to: double antibody sandwich ELISA method or any other immunoblotting method.

[0031] In a preferred embodiment of the present invention, the above evaluation of tumor treatment effect includes: if the PPIB level after tumor treatment is lower than the PPIB level before tumor treatment, the tumor treatment is evaluated to be effective; specifically, it is also manifested as tumor shrinkage.

[0032] If the PPIB level after tumor treatment is higher than the PPIB level before tumor treatment, it is evaluated that the tumor treatment effect is poor.

[0033] In a preferred embodiment of the present invention, the above evaluation of tumor treatment effect includes: if in multiple tumor treatment cycles, the PPIB level in the later stage of treatment is higher than the PPIB level in the early stage of tumor treatment, it is assessed that the tumor may have drug resistance and the treatment plan needs to be adjusted.

[0034] In a preferred embodiment of the present invention, in the above evaluation of tumor treatment effect, the sample to be tested is body fluid or exosomes. Exosomes are, for example, exosomes that have been processed by size exclusion chromatography, density gradient centrifugation, differential centrifugation, ultrafiltration, affinity capture, affinity purification, microfluidic separation, or a combination thereof.

[0035] In a preferred embodiment of the present invention, the body fluid is selected from urine, serum, plasma, peripheral blood and umbilical cord blood.

[0036] The present invention has the following beneficial effects:

[0037] The present invention provides a new use of PPIB protein as a protein marker for evaluating the therapeutic effect of tumors, which can be used for monitoring the therapeutic effect of tumors and for prognosing recurrence. Compared with existing protein markers, PPIB protein has higher sensitivity and specificity for patient treatment monitoring and prognosis. By detecting or monitoring the level of PPIB protein in patients, non-invasive tumor diagnosis can be achieved, which is of great significance for tumor treatment monitoring and recurrence prognosis, improving the effectiveness of treatment decisions, and prolonging patient survival time. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0039] Figure 1 This is the statistical result of the difference in serum CypB levels of 13 patients;

[0040] Figure 2 ROC curve diagram for evaluating PD and PR;

[0041] Figure 3 The ELISA results were for 41 cases;

[0042] Figure 4 This is a statistical result diagram of TCGA public data analysis. DETAILED DESCRIPTION

[0043] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0044] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0045] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0046] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0047] Example 1

[0048] This example evaluates the disease in 13 patients with lung cancer.

[0049] Before chemotherapy or before the next chemotherapy cycle, peripheral blood was collected from the patient and serum was separated. After the chemotherapy cycle was completed, peripheral blood was collected from the patient and serum was separated.

[0050] ELISA was used to detect the expression level of serum CypB:

[0051] (1) Dilute the standard (pure PPIB protein) according to the concentration gradient, dilute the test serum at 1:10, and dilute 8 gradients, namely 100 ng / ml, 40 ng / ml, 16 ng / ml, 6.4 ng / ml, 2.560 ng / ml, 1.024 ng / ml, 0.410 ng / ml, 0 ng / ml.

[0052] (2) Add 100ul of standard and test serum to the ELISA microplate coated with primary antibody ( ELH-CYPB ELISA kit). Incubate at 37°C for 2 h.

[0053] (3) Aspirate the standard and serum, add 300ul of washing solution to each well, shake the microplate gently, and then discard the washing solution. Repeat the above washing process three times.

[0054] (4) Add 100ul of diluted secondary antibody to each well ( ELH-CYPB ELISA kit) and incubated at 37°C for 2 h.

[0055] (5) Aspirate the standard and serum, add 300ul of washing solution to each well, shake the microplate gently, and then pour out the washing solution. Repeat the above washing process three times.

[0056] (6) Add diluted HRP (horseradish peroxidase) labeled secondary antibody and incubate at 37 degrees for 45 minutes.

[0057] (7) Absorbing HRP.

[0058] (8) Add 100 μl of color developing solution and develop the color at room temperature for 20 min.

[0059] (9) Add 50ul stop solution and read the OD value on the microplate reader. 480 nm wavelength detection readings.

[0060] (10) A standard curve was drawn based on the OD value to calculate the concentration of CypB in serum.

[0061] When used for prognostic treatment assessment, the assessment value = CypB 治疗后的水平 -CypB 治疗前的水平If the evaluation value is <0, that is, the CypB level decreases after treatment, it is predicted that the patient's treatment is effective, the tumor shrinks, and the disease is relieved. If the evaluation value is >0, that is, the CypB level increases after treatment, it is predicted that the patient's prognosis is poor and the treatment plan is ineffective.

[0062] When used for efficacy monitoring:

[0063] The patient's serum CypB level before the first treatment and at the end of each treatment cycle was recorded in sequence, and the difference in CypB levels between each treatment cycle was calculated (late minus early). When the evaluation value turns from negative to positive, it indicates that the tumor has begun to progress at the molecular level and may have begun to resist the drug. The treatment plan should be adjusted in time.

[0064] The inventors tested the CypB levels in the peripheral blood serum of 13 patients with Figure 1 As shown, the results show that 4 patients had an assessment value > 0, which was judged as disease progression, and predicted poor prognosis and poor treatment effect. 4 patients had an assessment value < 0, which predicted that the patient's treatment was effective, the tumor shrank, and the disease was relieved. 5 patients had stable disease (SD). The abbreviations PD = progressive disease, SD = stable disease (i.e., the tumor has not increased or shrunk to 15% of its original volume on imaging), and PR = disease remission.

[0065] The inventors further used the relationship between the above CypB evaluation value and the disease status to evaluate the ROC curve of disease progression or remission, such as Figure 2 As shown. The results show that the evaluation method provided by the present invention has high evaluation accuracy and sensitivity. The abbreviations used are PD = progressive disease, SD = stable disease (i.e., the tumor has not increased or shrunk to 15% of its original volume on imaging at present), and PR = disease remission.

[0066] The inventors tested the CypB levels in the peripheral blood serum of 41 patients with non-small cell lung cancer, whose treatments included surgery, chemotherapy, and targeted therapy. Figure 3 As shown, patients were grouped according to their CypB levels and their survival time was found to be different.

[0067] Since the average median survival time of patients corresponding to different treatment methods is different, we are worried that there may be bias in directly comparing the survival time of patients. Therefore, according to the median survival time of the treatment method, patients are marked as having a long survival period (DFS greater than 1 year after surgery and PFS greater than 6 months after chemotherapy) and a short survival period.

[0068] Reference Figure 3 As shown in the figure, by comparing the serum CypB levels of the two groups of patients, it was found that the patients with short survival had higher CypB levels than those with long survival. That is, CypB can still predict the treatment prognosis of patients.

[0069] The inventors further used the resources of the tumor public sequencing database to analyze the difference between the patient's CypB expression and the survival prognosis after chemotherapy. Figure 4 As shown, it was found that patients with high CypB expression had a short survival and poor prognosis.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a reagent for detecting protein marker levels in the preparation of a detection product for prognosis assessment of tumor treatment patients, characterized in that: The protein marker is PPIB, the tumor treatment is chemotherapy, and the tumor is selected from non-small cell lung cancer.

2. The use according to claim 1, characterized in that: The product is selected from a reagent, a kit or a chip.

3. The use according to claim 1, characterized in that: The sample to be tested is serum.

Images