A kit for predicting the efficacy of immunotherapy for prostate cancer patients and application thereof

Abstract

The application discloses a kit for predicting the curative effect of immunotherapy on prostate cancer patients, which comprises reagent A, reagent B, reagent C, reagent D, reagent E and reagent F; the reagent A is a PBS buffer solution; the reagent B is a lymphocyte separation medium; the reagent C is a red blood cell lysis solution; the reagent D is a cell surface CD3 flow antibody; the reagent E is a cell surface CD19 flow antibody; and the reagent F is a 1% polyformaldehyde solution in terms of mass percentage concentration. The application further discloses an application method of the kit, which comprises the following steps: collecting venous blood of prostate cancer patients; extracting peripheral blood mononuclear cells (PBMC) in the venous blood of the prostate cancer patients; incubating the flow antibody; and performing flow cytometry analysis. The kit and the application thereof are simple and fast, non-invasive, repeatable, low in cost and short in detection time.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a reagent kit and its application for predicting the efficacy of immunotherapy in prostate cancer patients. Background Technology

[0002] Recent studies have shown that immunotherapy can provide durable survival benefits for advanced prostate cancer. However, the efficacy of immunotherapy varies among patients; some benefit from it, while others experience initial treatment resistance. Predicting and screening patients who may benefit from immunotherapy allows for early intervention, the development of appropriate treatment plans to improve prognosis, and effectively reduce treatment costs. How to predict and screen patients who may benefit from immunotherapy remains a major challenge in clinical practice.

[0003] To address these issues, researchers in the field have made significant efforts. Various kits for predicting the efficacy of immunotherapy in prostate cancer patients have been proposed. Existing kits primarily rely on tumor cell PD-L1 expression levels, microsatellite instability (high) / DNA mismatch repair deficiency, and tumor mutational burden (TMB) as predictive biomarkers. However, PD-L1's predictive accuracy is insufficient, and its negative predictive value is low. For example, some studies on gastric or gastroesophageal junction adenocarcinoma and urothelial carcinoma have shown no correlation between PD-L1 expression and ICI response rate or survival; among FDA-approved ICIs, only 28.9% use PD-L1 as a predictive biomarker for treatment prognosis. Prostate cancer has a low level of immune infiltration, and existing clinical trial results cannot clearly evaluate the value of PD-L1 in predicting the efficacy of prostate cancer immunotherapy; in the large-scale multi-cohort KEYNOTE-199 trial, the ORR for the PD-L1 positive and negative cohorts were 5% and 3%, respectively, with no statistically significant difference between the two. Invasive tissue biopsies are required, but these are not routinely performed on patients with advanced prostate cancer. The MSI-H / dMMR molecular phenotype is uncommon in prostate cancer; both tissue and liquid biopsies indicate a detection rate of only about 3% in prostate cancer. Detecting MSI-H / dMMR through high-throughput sequencing is prohibitively expensive. TMB naturally varies among different tumor types, with significant differences in TMB threshold values ​​between various tumors. While TMB has predictive value for immunotherapy efficacy in other cancers, it has not been found to be associated with immunotherapy efficacy in prostate cancer. TMB requires genetic testing analysis, which is costly.

[0004] The inventors' latest research found that patients who benefited from immunotherapy had a lower percentage of CD3-negative and CD19-negative lymphocytes in their peripheral blood compared to those who did not. Furthermore, prospective cohort validation revealed that the percentage of CD3-negative and CD19-negative lymphocytes in peripheral blood can predict the efficacy of immunotherapy for prostate cancer; patients with a lower percentage had better efficacy, while those with a higher percentage had poorer efficacy. Based on this discovery, clinicians can screen patients for those who will benefit from and will not benefit from immunotherapy before treatment, thus determining whether immunotherapy should be used. This invention aligns with the principles of modern precision medicine and provides a new direction for personalized treatment of prostate cancer patients. Summary of the Invention

[0005] The main objective of this invention is to provide a kit and its application for predicting the efficacy of immunotherapy in prostate cancer patients. This kit and its application demonstrate good predictive ability for immunotherapy efficacy in advanced prostate cancer. It predicts the efficacy of immunotherapy by measuring the percentage of CD3-negative and CD19-negative lymphocytes in the peripheral blood of patients with advanced prostate cancer. It requires only 5 ml of peripheral venous blood from the patient, eliminating the need for a biopsy, making it a simple, rapid, non-invasive, and repeatable predictive method. The cost of this kit and its application is significantly lower than current immunotherapy efficacy prediction technologies, offering both low cost and short detection time.

[0006] To achieve the above objectives, the technical solution adopted by this invention is as follows: a kit for predicting the efficacy of immunotherapy in prostate cancer patients, characterized in that it includes reagents A, B, C, D, E, and F; reagent A is PBS buffer (phosphate buffered saline solution), which, as a buffer, has salt balance and an adjustable pH buffering effect; reagent B is a lymphocyte separation solution; reagent C is a erythrocyte lysis solution, which utilizes the principle of cell membrane rupture caused by the concentration difference of salt ions inside and outside the cell to lyse anucleate erythrocytes, mainly used for the removal of erythrocytes in lymphocyte separation and purification experiments; reagent D is a cell surface CD3 flow cytometry antibody, used to bind to CD3 on the surface of lymphocytes, and then analyze the proportion of CD3 positive cells by flow cytometry; reagent E is a cell surface CD19 flow cytometry antibody, used to bind to CD19 on the surface of lymphocytes, and then analyze the proportion of CD19 positive cells by flow cytometry; reagent F is a 1% (w / w) paraformaldehyde solution, used to fix the original morphological structure of lymphocytes.

[0007] Another object of the present invention is to provide a method for applying the aforementioned kit for predicting the efficacy of immunotherapy in prostate cancer patients, characterized by comprising the following steps:

[0008] Step S101: Collect venous blood from a prostate cancer patient;

[0009] Step S102: Extract peripheral blood mononuclear cells (PBMCs) from venous blood of prostate cancer patients.

[0010] Step S103: Incubate flow cytometry antibody;

[0011] Step S104: Perform flow cytometry analysis;

[0012] Preferably, the specific steps for extracting peripheral blood mononuclear cells (PBMCs) from the venous blood of prostate cancer patients in step S102 include:

[0013] Step S1021: Collect peripheral blood from the patient to be tested into an EDTA anticoagulant tube;

[0014] Step S1022: Add reagent A, dilute and mix well;

[0015] Step S1023: Take a 15ml centrifuge tube with the same number of samples and add reagent B: 3ml of lymphocyte separation solution;

[0016] Step S1024: Add 2-3 drops of blood sample, wait 10 seconds for it to form a film, and then slowly add the entire blood sample along the wall of the centrifuge tube;

[0017] Step S1025: Centrifugation;

[0018] Step S1026: After centrifugation, the blood separates into 4 layers. Remove the upper layer of liquid except for the red blood cells at the bottom.

[0019] Step S1027: Add reagent A, fill to the top, 1500 rpm, 5 min;

[0020] Step S1028: Add reagent C, 5 ml, mix well, incubate for 7 min to remove red blood cells;

[0021] Step S1029: Add reagent A to stop the lysis of red blood cells;

[0022] Step S10210: Centrifuge at 1500 rpm for 5 min and remove the supernatant;

[0023] Step S10211: Add 2ml of reagent A, resuspend the cell pellet, and obtain the patient's PBMCs.

[0024] Preferably, the centrifugation speed in step S1025 is 400g, the centrifugation temperature is 4℃, and the centrifugation time is 25~30min.

[0025] Preferably, the specific steps for incubating the flow cytometry antibody in step S103 include:

[0026] Step S1031: Take the EP tube and label it with the sample name;

[0027] Step S1032: Add reagent D and reagent E, and incubate in the dark for 25 min;

[0028] Step S1033: Add reagent A, fill to the top, mix well, incubate at 1500 rpm for 5 min, then discard the supernatant;

[0029] Step S1034: Add reagent A, resuspend the precipitate by pipetting, 1500 rpm, 5 min, and discard the supernatant;

[0030] Step S1035: Add 200 μL of reagent A to each tube, resuspend the precipitate by pipetting, and immediately proceed with 4. Flow cytometry analysis;

[0031] Step S1036: If flow cytometry analysis cannot be performed in time, fix the sample; add 200 μL of reagent F to each tube, resuspend the precipitate by pipetting, and store in a light-protected environment at 4°C.

[0032] Preferably, the flow cytometry analysis in step S104 specifically includes:

[0033] Step S1041: Delineate lymphocytes according to the conventional gating rules of human immunophenotype;

[0034] Step S1042: Determine the proportion of CD3-negative and CD19-negative cells in lymphocytes based on the differential expression of CD3 and CD19;

[0035] Step S1043: Based on the 30% threshold, predict the efficacy of immunotherapy for patients.

[0036] Preferably, the criteria for predicting the efficacy of immunotherapy in step S1043 are as follows: if the proportion of CD3-negative and CD19-negative cells in lymphocytes is greater than 30%, the predicted efficacy of immunotherapy may be poor, and immunotherapy is not recommended for the patient; if the proportion of CD3-negative and CD19-negative cells in lymphocytes is less than or equal to 30%, the predicted efficacy of immunotherapy may be good, and immunotherapy is recommended for the patient.

[0037] Due to the application of the above-mentioned technical solution, the present invention has the following advantages compared with the prior art:

[0038] (1) The kit for predicting the efficacy of immunotherapy in prostate cancer patients provided by the present invention predicts the efficacy of immunotherapy in prostate cancer by the proportion of CD3-negative and CD19-negative lymphocytes in peripheral blood. The prediction accuracy is high, which can enable early intervention for patients, timely and personalized symptomatic treatment, and the formulation of reasonable treatment plans to improve prognosis and effectively reduce the treatment cost for patients.

[0039] (2) The kit for predicting the efficacy of immunotherapy in prostate cancer patients provided by the present invention includes reagent A, reagent B, reagent C, reagent D, reagent E and reagent F; through the cooperation of each reagent, the kit can accurately predict the efficacy of immunotherapy in prostate cancer patients by only drawing 5ml of peripheral venous blood from the patient during the process, without the need for a biopsy of the patient; it is non-invasive, repeatable and has good detection effect; and it is low in cost.

[0040] (3) The application of the kit for predicting the efficacy of immunotherapy in prostate cancer patients provided by the present invention includes the following steps: taking venous blood from prostate cancer patients; extracting peripheral blood mononuclear cells (PBMCs) from the venous blood of prostate cancer patients; incubating flow cytometry antibodies and performing flow cytometry analysis. It requires few reagents, is easy to operate, and can safely, simply and quickly predict the efficacy of immunotherapy in prostate cancer patients, so as to realize targeted and personalized treatment of prostate cancer, improve the cure rate, reduce the mortality rate of patients, reduce the economic burden of patients, reduce the physical pain of patients, and improve the quality of life of patients.

[0041] (4) The application of the kit for predicting the efficacy of immunotherapy in prostate cancer patients provided by the present invention uses flow cytometry to delineate the proportion of CD3-negative and CD19-negative cells in lymphocytes based on the differential expression of CD3 and CD19. This effectively avoids the shortcomings of traditional methods, such as insufficient prediction accuracy, low negative predictive value, low level of immune infiltration, and high cost due to the need for gene detection analysis. Through creative work, a 30% threshold is derived to predict the efficacy of immunotherapy in patients. This results in low prediction cost, high prediction accuracy, convenient operation and use, no deficiency of low level of immune infiltration, no need for gene detection, and suitability for large-scale use. It has good clinical application results. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the reagent kit structure involved in the present invention.

[0043] Figure 2 This is a flowchart illustrating the application of the reagent kit for predicting the efficacy of immunotherapy in prostate cancer patients, as described in this invention.

[0044] Figure 3 This refers to the delineation of lymphocyte subsets in the patient's PBMC in Example 2 of the present invention.

[0045] Figure 4 This represents the proportion of CD3-negative and CD19-negative cells delineated in lymphocytes in Example 2 of this invention. Detailed Implementation

[0046] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0047] Example 1

[0048] A kit for predicting the efficacy of immunotherapy in prostate cancer patients, such as Figure 1 As shown, the reagents include reagents A, B, C, D, E, and F. Reagent A is PBS buffer (phosphate buffer solution), which serves as a buffer with salt balance and an adjustable pH buffering effect. Reagent B is a lymphocyte separation solution. Reagent C is a red blood cell lysis solution, which utilizes the principle of cell membrane rupture caused by the concentration difference of salt ions inside and outside the cell to lyse enucleated red blood cells, mainly used for the removal of red blood cells in lymphocyte separation and purification experiments. Reagent D is a cell surface CD3 flow cytometry antibody, used to bind to CD3 on the surface of lymphocytes, and then analyze the proportion of CD3-positive cells by flow cytometry. Reagent E is a cell surface CD19 flow cytometry antibody, used to bind to CD19 on the surface of lymphocytes, and then analyze the proportion of CD19-positive cells by flow cytometry. Reagent F is a 1% (w / w) paraformaldehyde solution, used to fix the original morphological structure of lymphocytes.

[0049] Example 2

[0050] In a case of metastatic castration-resistant prostate cancer (mCRPC), the peripheral blood immune cell subset ratio analysis of this invention was used to predict treatment efficacy, including:

[0051] (1) Collect 5ml of peripheral blood.

[0052] (2) Immune cells were isolated and extracted using the kit of the present invention, and flow cytometry antibodies were incubated to prepare the sample to be tested.

[0053] (3) Flow cytometry was used for analysis to obtain data ( Figure 3 ,4), the proportion of CD3-negative and CD19-negative cells in the patient's lymphocytes was 58.1%.

[0054] (4) Based on the 30% threshold, the proportion of CD3-negative and CD19-negative cells in the patient's lymphocytes is 58.1%, which is greater than the efficacy prediction threshold of 30%. It is predicted that the patient may have poor efficacy of immunotherapy and is not recommended to receive immunotherapy.

[0055] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A kit for predicting the efficacy of immunotherapy in prostate cancer patients, characterized in that, The reagents include reagent A, reagent B, reagent C, reagent D, reagent E, and reagent F; reagent A is PBS buffer, which has salt balance and an adjustable pH buffering effect; reagent B is lymphocyte separation solution. Reagent C is a red blood cell lysis buffer, which utilizes the principle of cell membrane rupture caused by the concentration difference of salt ions inside and outside the cell to lyse enucleated red blood cells. It is mainly used for the removal of red blood cells in lymphocyte isolation and purification experiments. Reagent D is a cell surface CD3 flow cytometry antibody, which is used to bind to CD3 on the surface of lymphocytes, and then analyze the proportion of CD3 positive cells by flow cytometry. Reagent E is a cell surface CD19 flow cytometry antibody, which is used to bind to CD19 on the surface of lymphocytes, and then analyze the proportion of CD19 positive cells by flow cytometry. Reagent F is a 1% (w / w) paraformaldehyde solution, which is used to fix the original morphological structure of lymphocytes.

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