Use of tfh cell subset in evaluation of r-chop treatment effect in dlbcl patient

Abstract

The use of a Tfh cell subset in the evaluation of the R-CHOP treatment effect in a DLBCL patient and the use of a reagent for detecting a Tfh cell subset in the preparation of a product for evaluating the R-CHOP treatment effect in a DLBCL patient, wherein the Tfh cell subset is a cell subset at the protein level with a cell surface marker defined as CD4+CXCR5+PD-1-; and the higher the proportion of CD4+CXCR5+PD-1-Tfh cells, the better the prognostic effect for a patient, and the longer the survival time of the patient. The CD4+CXCR5+PD-1-Tfh cells can be used for evaluating the R-CHOP treatment effect and prognosis for DLBCL.

Description

Application of Tfh cell subsets in evaluating the efficacy of R-CHOP treatment in patients with DLBCL Technical Field

[0001] The present invention relates to the technical field of tumor prognosis assessment, and in particular to the application of Tfh cell subsets in evaluating the efficacy of R-CHOP treatment in DLBCL patients. Background Art

[0002] Diffuse large B cell lymphoma (DLBCL) is the most common and prevalent subtype of non-Hodgkin lymphoma (NHL), accounting for 30% to 35% of all NHL cases. DLBCL is a heterogeneous group of tumors with significant variations in clinical presentation, histomorphology, and prognosis. The advent of rituximab has increased the cure rate for DLBCL to 60% to 80%, but one-third of patients still have a poor prognosis. Early identification of patients with a poor prognosis and timely adjustment of treatment strategies to prolong survival are crucial.

[0003] Advances in molecular pathology and targeted therapy have revealed numerous prognostic factors for DLBCL. Currently, clinically available prognostic factors for survival include the International Prognostic Index (IPI) score, MYC and BCL-2 expression, cell of origin, gene mutations, genetic risk prediction models, circulating tumor DNA (ct-DNA), the ratio of the absolute lymphocyte count to the absolute monocyte count (ALC / AMC) in peripheral blood, programmed death-1 ligand (PD-L1), and serum soluble interleukin-2 receptor (sIL-2R). However, most of these indicators lack independent validation, limiting their clinical use. The most widely used and guideline-endorsed prognostic scoring system is the IPI, which has been widely used for prognosis assessment in patients with aggressive NHL since 1993. However, in recent years, with the continuous progress of targeted immunotherapy, rituximab combined with chemotherapy has improved the prognosis of DLBCL patients, and the status of the IPI score in the prognostic system has been continuously impacted. Some scholars have proposed a modified international prognostic index (National Comprehensive Cancer Network-international prognostic index, NCCN-IPI) and GELTAMO-IPI (G-IPI) stratification method based on the IPI score. Compared with the IPI, it has higher predictive efficiency, but it still cannot fully identify high-risk groups. The IPI and its derived scoring systems provide patients with simple and reliable prognostic information at the time of diagnosis and are the benchmark for measuring all additional prognostic factors. However, the IPI has not had a substantial impact on treatment so far. There is currently no data to support changes in treatment strategies based on the IPI score, and it cannot predict responses to new drug treatments.

[0004] Therefore, there is an urgent need to establish a more complete DLBCL prognosis assessment system to truly divide high-risk groups, while fully utilizing the advantages of targeted therapy, adopting the most appropriate treatment plan to improve patient remission rates, reduce drug resistance and prolong patient survival, and guide clinical physicians to give patients more precise individualized treatment.

[0005] Summary of the Invention

[0006] In view of this, an object of the present invention is to provide the use of Tfh cell subsets in evaluating the efficacy of R-CHOP treatment in DLBCL patients or screening clinical therapeutic drugs; a second object of the present invention is to provide the use of reagents for detecting Tfh cell subsets in the preparation of products for evaluating the efficacy of R-CHOP treatment in DLBCL patients.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. Application of Tfh cell subsets in evaluating the efficacy of R-CHOP treatment in DLBCL patients, characterized in that the Tfh cell subsets are defined as CD4 + CXCR5 + PD-1 - cell subpopulations.

[0009] Tfh cell subsets CD4 + CXCR5 + PD-1 - The higher the proportion in the total cells, the better the R-CHOP treatment efficacy and the longer the prognosis and survival of DLBCL patients.

[0010] 2. The use of reagents for detecting Tfh cell subsets in the preparation of products for evaluating the efficacy of R-CHOP treatment in DLBCL patients.

[0011] Preferably, the reagent for detecting Tfh cell subsets includes one or more immune reagents of CD3E, CD3D, CD3G, CD4, BCL6, CXCR5, EBI3, NMB, IFNG or PDCD1.

[0012] Preferably, the immunoreagent is used for immunofluorescence / immunohistochemistry antibody or immunocolloidal gold labeling.

[0013] Preferably, the immune reagent includes a colorimetrically labeled molecular probe that specifically recognizes CD20, CD4, CXCR5, and PD-1.

[0014] Preferably, the color developer-labeled molecular probes are CD20 molecular probes, CD4 molecular probes, CXCR5 molecular probes, and PD-1 molecular probes labeled with different fluorescent markers, respectively.

[0015] Preferably, the product of the present invention further comprises a dewaxing agent, a gradient ethanol solution, a blocking solution, a dye, a DAPI stain and a TBST buffer.

[0016] The present invention preferably detects Tfh cell subsets CD4 + CXCR5 + PD-1- The higher the proportion in the total cells, the better the R-CHOP treatment efficacy and the longer the prognosis and survival of DLBCL patients.

[0017] The beneficial effects of the present invention are as follows: the present invention discloses the application of Tfh cell subsets in evaluating the efficacy of R-CHOP treatment in DLBCL patients. The present invention uses single-cell RNA sequencing technology, external database data and multiple immunofluorescence (mIF) detection technology to analyze the characteristics and spatial distribution characteristics of new follicular helper T cell (Tfh) subsets in DLBCL and immune microenvironment, and finds that in tumor samples of relapsed patients, the proportion of Tfh cell subsets with high expression of CD3E, CD3D, CD3G, CD4, BCL6, CXCR5, EBI3 or NMB and low expression of IFNG or PDCD1 genes is significantly reduced, suggesting that this Tfh cell subset may be related to poor tumor prognosis. Further in-depth analysis of the CD4 follicular helper T cell (Tfh) subsets in the microenvironment of tumor tissue samples of DLBCL patients who respond and do not respond to R-CHOP treatment was conducted. + CXCR5 + PD-1 - The proportion of tumor cells interacts with the space and CD4 + CXCR5 + PD-1 - The higher the proportion of Tfh cells, the better the prognosis of the patient and the longer the survival period. + CXCR5 + PD-1 - Tfh cells can be used to evaluate the efficacy and prognosis of R-CHOP treatment for DLBCL, and have great application prospects in biological fields and medical translational research. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0019] Figure 1 is a flowchart of sequencing library data analysis;

[0020] Figure 2, A is a UMAP analysis of immune cell subsets using scRNA-seq in DLBCL tumor tissue; B is a gene annotation diagram of cell subsets; C is a heat map of differentially expressed genes in different subpopulations; D is a heat map of T cell subset definitions and differentially expressed genes;

[0021] Figure 3, A is the relationship between Tfh cell subsets and poor tumor prognosis; B is the relationship between the proportion of Tfh cell subsets and long-term tumor prognosis, q1, q2, q3, and q4 represent different regional segments, and the quartiles represent 25%, 50%, 75%, and 100%, respectively;

[0022] Figure 4, A is the image of tissue staining and CD4 + CXCR5 + PD-1 - Tfh cell marker image; B is CD4 + CXCR5 + PD-1 - The proportion of Tfh cells in the total cells was higher in the R-CHOP response group than in the R-CHOP non-response group; C is CD4 + CXCR5 + PD-1 - The proportion of Tfh cells in total cells and prognostic analysis;

[0023] Figure 5, A is CD4 + CXCR5 + PD-1 - Analysis of the spatial distribution of Tfh cells and their interaction with tumor cells; B is CD4 + CXCR5 + PD-1 - Distribution of Tfh cells in R-CHOP response and R-CHOP non-response patient groups; C is CD4 + CXCR5 + PD-1 - Relationship between Tfh cell distribution and prognosis of DLBCL patients. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0025] Example 1

[0026] 1. Dissociate fresh DLBCL tumor tissue samples to isolate single cell suspensions. The specific steps are as follows:

[0027] (1) After surgery, the excised tumor tissue was immersed in PBS and moved to a clean bench. The tissue was cut into 5 mm pieces using a sterile scalpel. 3 Size: about the size of a soybean;

[0028] (2) Wash the minced tissue 1-2 times with PBS and store the tissue in tissue protection solution (130-100-008, Miltenyi Biotec), making sure that the sample is submerged in the protection solution;

[0029] (3) Refer to the tissue dissociation kit (130-095-929, Miltenyi Biotec) operating procedures to dissociate tumor tissue into single-cell suspension; sample requirements: cell viability ≥ 90%, uniform cell size with a diameter between 7-40 μm, cell clusters and debris impurities < 5%, double cell rate < 0.09%, cell number ≥ 100,000, and no reverse transcription inhibitors are present in the cell suspension.

[0030] 2. Library construction and sequencing. The specific steps are as follows:

[0031] (1) 10x single-cell RNA sequencing technology labels cDNA fragments. Gel beads containing barcode information are first bound to a mixture of cells and enzymes, and then encapsulated by oil-surfactant droplets located in a microfluidic "double cross" connection. The droplets are very small and can only accommodate one gel bead. After the droplets flow into the reservoir and are collected, the gel beads dissolve and release the primer sequence, reverse transcribe the cDNA fragments, and perform PCR amplification using the cDNA as a template. The products containing barcode information in each droplet are mixed to construct a sequencing library.

[0032] (2) Library construction: First, the cDNA is broken into fragments of about 200-300 bp, and then the sequencing adapter P5 and sequencing primer R1 are added to the traditional second-generation sequencing library construction process. Finally, PCR amplification is performed to obtain the DNA library.

[0033] (3) Cluster generation and sequencing: The library is quantified and qualified libraries are placed in a cBot for bridge amplification. After cluster generation, sequencing is performed using a sequencer. The principle is that dNTPs labeled with four fluorescent groups, A, T, G, and C, are added in each cycle. Based on the AT and GC pairing, the corresponding dNTPs are bound to the template DNA chain by DNA polymerase, and other unbound dNTPs are washed away. The binding position releases a fluorescent signal, which is captured by the computer and converted accordingly, thereby obtaining the base information at that position.

[0034] 3. The data analysis process is shown in Figure 1, and is verified by combining sample clinical information and survival data from external databases.

[0035] First, a total of 9 fresh DLBCL tumor tissue samples, both newly diagnosed and relapsed, were collected from Sichuan Cancer Hospital. Using single-cell RNA sequencing, scRNA-seq immune cell subset UMAP analysis was performed on the DLBCL tumor tissues (Figure 2, A). Gene annotation of the cell subsets was performed (Figure 2, B). A heat map of differentially expressed genes across the subpopulations was shown in Figure 2, C. T cell subset definitions and a heat map of differentially expressed genes were shown in Figure 2, D. Reference genes for single-cell RNA sequencing of the new Tfh cell subpopulation are shown in Table 1.

[0036] Table 1. Reference genes for new Tfh cell subsets sequenced by single-cell sequencing

[0037] By analyzing single-cell sequencing data (an average of 8,000 to 10,000 cells were collected and tested per sample), it was found that in tumor samples from recurrent patients, the proportion of Tfh cell subsets with high expression of CD3E, CD3D, CD3G, CD4, BCL6, CXCR5, EBI3 or NMB and low expression of IFNG or PDCD1 genes decreased significantly, suggesting that this Tfh cell subset may be associated with poor tumor prognosis (Figure 3, A). Further large-sample verification and comparison by collecting RNA bulk sequencing data from newly diagnosed DLBCL tumor tissue samples from the external public database TCGA also found that in newly diagnosed DLBCL patients, the higher the proportion of secondary Tfh cell subsets in the tumor tissue microenvironment, the better the patient's long-term prognosis (Figure 3, B).

[0038] Example 2

[0039] Tumor tissue samples from DLBCL patients who responded to R-CHOP treatment (14 patients) and those who did not respond (18 patients) at Sichuan Cancer Hospital were obtained. Hematoxylin and eosin staining was performed to initially characterize the spatial regions of the tissues, group them, and conduct a follow-up cohort study. Tumor tissue samples were stained with multiple fluorescent markers for CD20, CD4, CXCR5, and PD-1, and with DAPI (Novo-Light TSA, Shanghai Yuanxi Biotechnology Co., Ltd.). Panoramic scanning of the sections was performed using a fluorescence imaging scanner (3DHISTECH). The specific steps were as follows:

[0040] (1) Dewaxing, antigen retrieval, and staining were performed using a Leica Bond III stainer. Antigen retrieval conditions were ER2 antigen retrieval solution at 100°C for 20 min. Wash with TBST 3-5 times after each step between (2)-(5).

[0041] (2) Blocking: 3% hydrogen peroxide (Peroxide Block) for 10 min, followed by blocking with blocking solution for 10 min at room temperature, and washing with TBST 3-5 times;

[0042] (3) Antibody dilution and staining: Wash 3-5 times with TBST after each step between the ab steps;

[0043] CD20:

[0044] a. Primary antibody incubation: Incubate with CD20 antibody stock solution at room temperature for 60 minutes.

[0045] b. Secondary antibody incubation: Incubate with Leica DS9800 staining system secondary antibody (Post Primary / Polymer) at room temperature for 10 min.

[0046] c. Incubate with fluorescent dye Novo-Light TSA620 at 1:100 at room temperature for 10 minutes.

[0047] PD-1:

[0048] a. Primary Antibody Incubation: Dilute PD-1 antibody in Antibody Diluent / Blocking Buffer at a concentration of 1:400 and incubate at room temperature for 60 minutes.

[0049] b. Secondary antibody incubation: Incubate with Leica DS9800 staining system secondary antibody (Post Primary / Polymer) at room temperature for 10 min.

[0050] c. Incubate with fluorescent dye Novo-Light TSA570 at 1:100 at room temperature for 10 minutes.

[0051] CXCR5:

[0052] a. Primary Antibody Incubation: Dilute CXCR5 antibody in Antibody Diluent / Blocking Buffer at a concentration of 1:200 and incubate at room temperature for 30 min.

[0053] b. Secondary antibody incubation: Incubate with Leica DS9800 staining system secondary antibody (Polymer) at room temperature for 10 min.

[0054] c. Incubate with fluorescent dye Novo-Light TSA 520 at a dilution of 1:100 at room temperature for 10 minutes.

[0055] CD4:

[0056] a. Primary Antibody Incubation: Dilute CD4 antibody in Antibody Diluent / Blocking Buffer at a concentration of 1:400 and incubate at room temperature for 30 minutes.

[0057] b. Secondary antibody incubation: Incubate with Leica DS9800 staining system secondary antibody (Polymer) at room temperature for 10 min.

[0058] (4) Incubate with DAPI for 10 minutes to stain the nucleus

[0059] (5) Seal the slides with anti-fluorescence quencher and take pictures.

[0060] In this example, the antibodies for markers CD20, CD4, CXCR5, and PD-1 are as follows:

[0061] The primary antibody for CD20 labeling was rabbit anti-human CD20 monoclonal antibody (CM-0221, Shanghai Jiehao Biotechnology Co., Ltd.), and the secondary antibody was Leica DS9800 staining system secondary antibody (Post Primary / Polymer).

[0062] The primary antibody for CD4 labeling was rabbit anti-human CD4 monoclonal antibody (ab133616, Abcam), and the secondary antibody was Leica DS9800 staining system secondary antibody (Post Primary / Polymer).

[0063] The primary antibody for CXCR5 labeling was rabbit anti-human CXCR5 monoclonal antibody (72712S, CST), and the secondary antibody was Leica DS9800 staining system secondary antibody (Post Primary / Polymer).

[0064] The primary antibody for PD-1 labeling was rabbit anti-human PD-1 monoclonal antibody (10377-MM23, Sino biological), and the secondary antibody was Leica DS9800 staining system secondary antibody (Post Primary / Polymer).

[0065] Tissue staining images and CD4 + CXCR5 + PD-1 - The iconic image of TFH cells is shown in Figure 4, A. The High plex FL module of the digital case analysis software Halo (Indica Lab) was used to classify single cells in the tissue, and the results showed that CD4 + CXCR5 + PD-1 - The proportion of Tfh cells in the total cells was higher in the R-CHOP response group than in the R-CHOP non-response group, which successfully predicted the effect of R-CHOP treatment in DLBCL patients. The results are shown in Figure 4, B. + CXCR5 + PD-1 - The prognostic analysis of the proportion of Tfh cells in the total cells showed that CD4 + CXCR5 + PD-1 - DLBCL patients with a high proportion of Tfh cells were successfully predicted to have a better prognosis, as shown in Figure 4, C.

[0066] The spatial analysis module of digital medical analysis software Halo (Indica Lab) was used to analyze CD4 + CXCR5 + PD-1 -The spatial distribution of Tfh cells and their interaction with tumor cells were analyzed, with 20 μm as the cell interaction distance (Figure 5, A). The results showed that CD4 + CXCR5 + PD-1 - Tfh cells were more abundant in the R-CHOP response group than in the R-CHOP non-response group, successfully predicting the effect of R-CHOP treatment in DLBCL patients (Figure 5, B); CD4 + CXCR5 + PD-1 - Prognostic analysis of Tfh cells showed that CD4 + CXCR5 + PD-1 - DLBCL patients with more Tfh cells had a better prognosis, and the prognosis of DLBCL patients was successfully predicted. The results are shown in Figure 5, C.

[0067] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. Application of Tfh cell subsets in evaluating the efficacy of R-CHOP treatment in DLBCL patients, characterized in that: The Tfh cell subset is defined as CD4 + CXCR5 + PD-1 - cell subpopulations.

2. The use according to claim 1, wherein the Tfh cell subset CD4 + CXCR5 + PD-1 - The higher the proportion in the total cells, the better the R-CHOP treatment efficacy of DLBCL patients and the longer the prognosis and survival period.

3. Application of reagents for detecting Tfh cell subsets in the preparation of products for evaluating the efficacy of R-CHOP treatment in DLBCL patients.

4. The use according to claim 3, characterized in that: The reagents for detecting Tfh cell subpopulations include one or more immune reagents of CD3E, CD3D, CD3G, CD4, BCL6, CXCR5, EBI3, NMB, IFNG or PDCD1.

5. The use according to claim 3, characterized in that: The immunological reagent is used for immunofluorescence / immunohistochemistry antibody or immunocolloidal gold labeling.

6. The use according to claim 4, characterized in that: The immune reagent includes a colorimetric marker molecular probe that specifically recognizes CD20, CD4, CXCR5 and PD-1.

7. The use according to claim 6, characterized in that: The color developer-labeled molecular probes are CD20 molecular probe, CD4 molecular probe, CXCR5 molecular probe and PD-1 molecular probe respectively labeled with different fluorescence.

8. The use according to claims 3-7, characterized in that: The product also includes a dewaxing agent, a gradient ethanol solution, a blocking solution, a dye, a DAPI stain, and a TBST buffer.

9. The use according to claim 3 to 7, wherein the Tfh cell subset CD4 + CXCR5 + PD-1 - The higher the proportion in the total cells, the better the R-CHOP treatment efficacy and the longer the prognosis and survival of DLBCL patients.

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