Marker for predicting non-small cell lung cancer and immune-related adverse events and application thereof

Abstract

The invention discloses a marker for predicting non-small cell lung cancer and immune-related adverse events and application thereof. The marker is CD8 < + > PD-1 < + > T. According to the proportion value of CD8 + PD-1 + T in CD8 + T, the occurrence risk and time of irAE larger than or equal to 2 of local advanced NSCLC in the ICIs-CRT + / -ICIs treatment mode can be effectively predicted. According to the CD8 + PD-1 + T-based noninvasive prediction method for predicting that the local advanced NSCLC is greater than or equal to level 2 irAE, the index detection method is simple, convenient and feasible, the prediction efficiency is high, and the generalizability is high. Great help is provided for diagnosis and treatment of lung cancer in China.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor detection, and in particular, relates to the use of a marker in constructing a model, a detection reagent, a detection kit or a device for predicting immune-related adverse events of locally advanced non-small cell lung cancer in an ICIs-CRT±ICIs treatment mode. Background Art

[0002] Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of lung cancer. Concurrent chemoradiotherapy (CRT) combined with consolidation ICIs has become the standard treatment for locally advanced non-small cell lung cancer (NSCLC). Despite receiving the standard treatment regimen of consolidation ICIs, the cure rate of locally advanced NSCLC is still low, with approximately 70% of patients progressing within 5 years. Given that ICIs can stimulate the strongest anti-tumor immune efficacy when the primary tumor and metastatic lymph nodes are intact, the new treatment model of induction chemoimmunity combined with chemoradiotherapy ± consolidation immunotherapy (ICIs-CRT±ICIs), which further advances ICIs to the induction therapy stage, has been explored by major research teams for its excellent treatment concept and cure potential. Preliminary research results show that advancing ICIs to the induction therapy stage may bring more potential benefits to cancer patients, but it is also accompanied by an increase in immune-related adverse events (irAEs). irAE can affect almost any organ in the body, and its frequency depends on the type of ICIs used, up to 70%. Symptomatic irAE (≥ grade 2) requires clinical intervention, of which 10-20% develop into severe irAE (≥ grade 3). Once severe irAE occurs, clinical symptoms may progress rapidly, and even life-threatening situations may occur. Therefore, finding risk predictors associated with symptomatic irAE and severe irAE will help screen patients at high risk of irAE and provide a reference for clinical treatment decisions.

[0003] Since the biological drivers of irAEs are still unclear and there is significant heterogeneity in ICIs-induced irAEs, finding accurate and reliable irAE-related biomarkers has always been a challenge.

[0004] At present, some studies have reported that peripheral blood immune cell subsets Ki-67+Tregs, Ki-67+CD8+T, effector memory CD4T and cytokines IFN-γ, CXCL9, CXCL10, CXCL11, etc. can predict irAEs in NSCLC patients to a certain extent, but the corresponding sample size is limited and the patient population targeted is stage IV NSCLC. Therefore, it is urgent to develop an accurate and reliable non-invasive predictive biomarker for irAEs in locally advanced NSCLC under the ICIs-CRT±ICIs treatment model. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a marker for use in constructing a model, a detection reagent, a detection kit or a device for predicting immune-related adverse events of locally advanced non-small cell lung cancer in the ICIs-CRT±ICIs treatment mode.

[0006] The present invention provides an application of a marker in constructing a model for predicting immune-related adverse events of locally advanced non-small cell lung cancer in an ICIs-CRT±ICIs treatment mode, wherein the marker is CD8 + PD-1 + T.

[0007] According to one embodiment of the present invention, the model includes a data acquisition module and a judgment and output module; the data acquisition module is used to obtain peripheral blood CD8 + PD-1 + T in CD8 + The proportion value of T; the judgment module is used to compare the proportion value with the threshold value to determine the result; the output module is used to output the judgment result obtained by the judgment module.

[0008] According to another embodiment of the present invention, the threshold is determined by peripheral blood CD8 + PD-1 + The relationship between the T ratio value and the outcome event ≥ grade 2 immune-related adverse events was obtained by AUC statistical analysis.

[0009] According to another embodiment of the present invention, the threshold is between 28.5% and 49.7%.

[0010] According to another embodiment of the present invention, if the CD8 + PD-1 + If the T ratio is greater than the threshold, the subject is in the high-risk group; + PD-1 +If the T ratio value is less than or equal to the threshold, the subject is in the low-risk group; alternatively, for subjects whose test value is greater than the threshold, the risk of experiencing grade ≥ 2 immune-related adverse events after induced immunization is greater than that for subjects whose test value is less than or equal to the threshold.

[0011] The present invention also provides a marker for use in the preparation of a detection reagent, a detection kit or a device for predicting immune-related adverse events of locally advanced non-small cell lung cancer in the ICIs-CRT±ICIs treatment mode, characterized in that the marker is CD8 + PD-1 + T.

[0012] According to one embodiment of the present invention, the detection reagent, detection kit or device includes anti-CD3-FITC (clone: ​​OKT3), anti-CD8-APC (clone: ​​RPA-T8), anti-CD279 (PD-1)-PE (clone: ​​EH12.2H7), and anti-IgG4Fc (clone: ​​HP6025).

[0013] According to another embodiment of the present invention, the application comprises: collecting a peripheral blood sample of an individual to be tested; adding a flow cytometry antibody to bind to a peripheral blood T cell surface antigen; using a flow cytometry sorter to detect a T cell suspension and obtain CD8 + PD-1 + T in CD8 + The proportion of T; and the risk and time of an individual experiencing ≥ grade 2 immune-related adverse events predicted based on the flow cytometer detection data and judgment threshold.

[0014] According to another embodiment of the present invention, the individual to be detected is a patient with locally advanced non-small cell lung cancer under ICIs-CRT±ICIs treatment mode.

[0015] The present invention provides a method based on CD8 + PD-1 + This is a non-invasive method for predicting ≥2 irAE in locally advanced NSCLC. The detection method of this indicator is simple and feasible, with high predictive efficiency and strong scalability. It is of great help to the diagnosis and treatment of lung cancer in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The gating strategy for flow cytometry is shown; in the figure, A represents the PD-1 isotype control, and B represents the PD-1 flow cytometry antibody.

[0017] Figure 2The competitive inhibition between anti-PD-1 ICIs and anti-PD-1 fluorescent antibodies is shown; A in the figure indicates that after treatment with toripalimab, anti-PD-1 fluorescent antibodies cannot fully recognize PD-1+T; B indicates that the addition of anti-IgG4 fluorescent antibodies can fully recognize PD-1+T; C indicates that after treatment with tislelizumab, anti-PD-1 fluorescent antibodies cannot recognize PD-1+T; D indicates that the addition of anti-IgG4 fluorescent antibodies can fully recognize PD-1+T.

[0018] Figure 3 The ROC curve of CD8+PD-1+T value predicting grade ≥2 irAE before consolidation ICIs is shown. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0020] The purpose of the present invention is to provide a predictive biomarker CD8 for irAE in locally advanced NSCLC in the ICIs-CRT±ICIs treatment model. + PD-1 + T. The application of this marker in constructing a model for predicting irAE in locally advanced NSCLC in the ICIs-CRT±ICIs treatment mode. It includes a data acquisition module, a judgment and output module; the data acquisition module is used to obtain peripheral blood CD8 + PD-1 + T accounts for CD8 + The proportion value of T; the judgment module is used to compare the proportion value with the threshold value to determine the result; the output module is used to output the judgment result obtained by the judgment module.

[0021] The threshold was determined by peripheral blood CD8 + PD-1 + The relationship between the T ratio value and the outcome event ≥ grade 2 irAE was obtained by AUC statistical analysis. In the present invention, the threshold value is between 28.5% and 49.7%.

[0022] If CD8 + PD-1 + The T ratio is greater than the threshold, and the subject is in the high-risk group. The risk of ≥ grade 2 irAE after induced immunity is high and the occurrence time is short. + PD-1 +If the T ratio value is less than or equal to the threshold, the subject is in the low-risk group, and the risk of the subject developing ≥ grade 2 irAE after induced immunity is low.

[0023] Alternatively, subjects whose test values ​​are greater than the threshold have a greater risk of developing grade ≥2 irAEs after induced immunity than subjects whose test values ​​are less than or equal to the threshold.

[0024] The above subjects are patients with locally advanced NSCLC.

[0025] The above-mentioned low-risk group and high-risk group are divided into subjects according to threshold values.

[0026] Another object of the present invention is to provide a biomarker CD8 + PD-1 + T detection reagents, detection kits or devices, including anti-CD3-FITC (clone: ​​OKT3), anti-CD8-APC (clone: ​​RPA-T8), anti-CD279 (PD-1)-PE (clone: ​​EH12.2H7), and anti-IgG4Fc (clone: ​​HP6025). Among them, CD3-FITC (clone: ​​OKT3), anti-CD8-APC (clone: ​​RPA-T8), anti-CD279 (PD-1)-PE (clone: ​​EH12.2H7) can be BioLegend brand, and anti-IgG4Fc (clone: ​​HP6025) is Southern Biotech brand. The present invention clearly proposes for the first time that there is competition for the epitopes of PD-1+T cells in peripheral blood after the use of anti-PD-1 ICIs, and it is necessary to add anti-CD279 (PD-1) flow cytometry antibodies and anti-IgG4Fc flow cytometry antibodies at the same time to fully identify PD-1. + T. This invention first proposed CD8 + PD-1 + T in CD8 + The percentage value of T can effectively predict the risk and time of grade ≥2 irAE in locally advanced NSCLC under the ICIs-CRT±ICIs treatment model.

[0027] In order to detect the biomarker CD8 in locally advanced NSCLC under ICIs-CRT±ICIs treatment mode + PD-1 +T, 86 patients with locally advanced NSCLC who received ICIs-CRT±ICIs were prospectively enrolled in three centers (NCC cohort: Cancer Hospital of Chinese Academy of Medical Sciences, independent validation cohort: Hengxing Cancer Hospital and Peking University Cancer Hospital). 4-10 ml of fresh peripheral blood was collected at four time points: baseline, before radiotherapy after induction of immunity, during radiotherapy (20f), and within 1 month after radiotherapy. Total leukocytes were obtained after lysis using lysis buffer (BD Bioscience). The leukocyte suspension was stained with human protein-specific flow cytometry antibodies at 4°C for 30 minutes. The antibodies used included BioLegend brand anti-CD3-FITC (clone: ​​OKT3), anti-CD8-APC (clone: ​​RPA-T8), and anti-CD279 (PD-1)-PE (clone: ​​EH12.2H7). For peripheral blood after anti-PD-1 drug treatment, Southern Biotech brand anti-IgG4Fc (clone: ​​HP6025) was added to recognize and bind to PD-1 in the leukocyte suspension. + CD8 + PD-1 + T detection was performed using FlowJo software (version 10.8.1) according to a predefined gating strategy to acquire CD8 + PD-1 + T data (such as Figure 1 shown).

[0028] Further investigation of CD8 + PD-1 + The method is as follows: (1) collect peripheral blood samples from the individual to be tested; (2) add flow cytometry antibodies to bind to peripheral blood T cell surface antigens; (3) use a flow cytometer to detect T cell suspension and obtain CD8 + PD-1 + (3) predicting the risk and time of an individual developing grade ≥ 2 irAE based on the flow cytometer detection data and judgment threshold.

[0029] The test individuals of the present invention are patients with locally advanced NSCLC under the ICIs-CRT±ICIs treatment mode.

[0030] The sample for predicting the risk and time of occurrence of grade ≥2 irAE in locally advanced NSCLC provided by the present invention is peripheral blood T cells. The peripheral blood T cell index is CD8 + PD-1 + The percentage of T cells in CD8+T cells. Further, the source of T cells is blood. Further, the detection product of the present invention is a reagent, a kit or a device.

[0031] Example 1

[0032] 1. Research ideas and patient conditions

[0033] The grade of irAE represents the severity of the adverse reaction. Grade ≥2 irAE is symptomatic and requires drug intervention. Therefore, this patent focuses on the predictive factors of grade ≥2 irAE. This patent collects clinical data and peripheral blood specimens according to the research method approved by the ethics committee, and obtains written informed consent from each patient in accordance with the Declaration of Helsinki.

[0034] The present invention prospectively enrolled patients with locally advanced NSCLC who received ICIs-CRT±ICIs in three centers (NCC cohort: Cancer Hospital of Chinese Academy of Medical Sciences, independent validation cohort: Hengxing Cancer Hospital and Peking University Cancer Hospital) from 2022 to 2024. 4-10 ml of fresh peripheral blood was collected at four time points: baseline, before radiotherapy after induction of immunity, during radiotherapy (20f), and within 1 month after radiotherapy for CD8 + PD-1 + Detection of T. Baseline demographic and treatment data were extracted from electronic medical records, including age, ECOG score, gender, smoking history, pathological type, eighth edition AJCC T NM tumor stage, PD-L1 expression level, ICIs treatment cycle and time, and other clinical data.

[0035] Hematological tests and physical examinations were performed during each anti-PD-1 / anti-PD-L1 antibody administration cycle. Imaging examinations were performed in the first month after CRT, then every 3 months in the first two years, and every 6 months thereafter. irAEs were graded according to the Common Terminology Criteria for Adverse Events Version (CTCAE) 5.0. The type and time of irAE occurrence were divided into three categories: no ≥ grade 2 irAEs, ≥ grade 2 irAEs before consolidation ICIs, and ≥ grade 2 irAEs after consolidation ICIs. The type of irAE was correlated with the patient's CD8 + PD-1 + The T values ​​were analyzed by receiver operating characteristic (ROC) curve. The CD8 + PD-1 + The T value range is the threshold range for judging ≥ grade 2 irAE.

[0036] 2. Patient characteristics

[0037] A total of 86 patients with locally advanced NSCLC who received ICIs-CRT±ICIs and had peripheral blood specimens collected were included in the analysis. Table 1 shows the characteristics of the enrolled patients, with 76.7% of the patients in the National Cancer Center cohort (n=66) and 23.2% of the patients in the independent validation cohort (n=20). The types of ≥2-grade irAEs and other clinical characteristics in the two groups were balanced, with no statistical differences.

[0038] Table 1 shows the clinical characteristics of the patients enrolled in the multicenter study

[0039]

[0040]

[0041] 3. Peripheral blood CD8 + PD-1 + Detection of T

[0042] 4-10 ml of fresh peripheral blood was collected and lysed with lysis buffer (BD Bioscience) to obtain total leukocytes. The leukocyte suspension was stained with human protein-specific flow cytometry antibodies for 30 minutes at 4°C. The antibodies used included BioLegend brand anti-CD3-FITC (clone: ​​OKT3), anti-CD8-APC (clone: ​​RPA-T8) and anti-CD279 (PD-1)-PE (clone: ​​EH12.2H7). Due to the competitive inhibition between anti-PD-1 ICIs and flow cytometry anti-PD-1 fluorescent antibodies, that is, PD-1 + The occupation of T cells by anti-PD-1 ICIs will affect the flow cytometry of anti-PD-1 fluorescent antibodies and PD-1 + T cell surface antigen binding, leading to PD-1 + The actual value of T is underestimated. Given that the anti-PD-1 ICIs currently available on the market are IgG4 in structure, and the IgG4 epitope on the surface of T cells is basically zero, therefore, for peripheral blood after anti-PD-1 drug treatment, anti-IgG4 Fc (clone: ​​HP6025) is added to recognize and bind to the antigen epitope in the leukocyte suspension that has bound to the PD-1 of anti-PD-1 ICIs. + cells, PD-1 + T and IgG4 + T is commonly defined as PD-1 + T(such as Figure 2 The stained cell suspension was sorted using the flow cytometry FACS Aria Fusion (BD Biosciences) for CD8 + PD-1 + T detection was performed using FlowJo software (version 10.8.1) according to a predefined gating strategy to acquire CD8+ PD-1 + T data, CD8 + PD-1 + The percentage of T is defined as CD8 + PD-1 + T / CD8 + The proportional value of T.

[0043] 4. CD8 + PD-1 + TThreshold range and performance for predicting grade ≥2 irAEs

[0044] Comparison of CD8 at each blood collection time point under different irAE type groups + PD-1 + T value, found that CD8 + PD-1 + The percentage of T was significantly higher than that of other patients. + PD-1 + ROC analysis was performed on the T value and the ≥2-grade irAE status before consolidation of ICIs. Figure 3 As shown in the figure, the AUC at the time point before radiotherapy reached 0.852, and the specificity, sensitivity and accuracy were 0.875, 0.750 and 0.842, respectively. + PD-1 + When the T value is in the range of 28.5 to 49.7, the Youden index is >0.4, which has good predictive efficiency; the highest AUC reaches 0.894. The independent analysis results of two different cohorts are similar, both suggesting that CD8 + PD-1 + T has high accuracy in predicting the time of ≥ grade 2 irAE before consolidation ICIs. Table 2 shows the time points before radiotherapy for different CD8 + PD-1 + The predictive efficacy of T-score for grade ≥2 irAEs.

[0045] Table 2

[0046]

[0047]

[0048]

[0049] Table 3 shows CD8 + PD-1 + Evaluation of the predictive efficacy of T for grade ≥2 irAE.

[0050] Table 3

[0051]

[0052]

[0053] Further adding the variable of immune medication time, we found that CD8 + PD-1 + The higher the T value, the shorter the time from the first immunotherapy to the occurrence of grade 2 irAE events. + PD-1 + The AUC of T reached 0.836.

[0054] Table 4 shows the prediction time efficacy evaluation of grade ≥2 irAE of CD8+PD-1+T.

[0055] Table 4

[0056] Time of occurrence of grade ≥2 irAE Specificity Sensitivity Accuracy <=1month 0.867 0.776 0.785 1-2month 0.778 0.843 0.836 2-5month 0.522 0.828 0.783 >5month 0.650 0.522 0.539

[0057] The present invention provides a method based on CD8 + PD-1 + This is a non-invasive method for predicting ≥2 irAE in locally advanced NSCLC. The detection method of this indicator is simple and feasible, with high predictive efficiency and strong scalability. It is of great help to the diagnosis and treatment of lung cancer in my country.

[0058] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Use of a marker in constructing a model for predicting immune-related adverse events in locally advanced non-small cell lung cancer in ICIs-CRT±ICIs treatment mode, characterized in that: The marker is CD8 + PD-1 + T.

2. The use according to claim 1, characterized in that: The model includes a data acquisition module, a judgment and output module; The data acquisition module is used to obtain peripheral blood CD8 + PD-1 + T in CD8 + The percentage value of T; The judging module is used to compare the percentage value with the threshold value to judge the result; The output module is used to output the judgment result obtained by the judgment module.

3. The use according to claim 2, characterized in that: The threshold was determined by peripheral blood CD8 + PD-1 + The relationship between the T ratio value and the outcome event ≥ grade 2 immune-related adverse events was obtained by AUC statistical analysis.

4. The use according to claim 3, characterized in that: The threshold value is between 28.5% and 49.7%.

5. The use according to claim 2, characterized in that: If the CD8 + PD-1 + If the T ratio value is greater than the threshold, the subject is in the high-risk group; If the CD8 + PD-1 + If the T ratio value is less than or equal to the threshold, the subject is in the low-risk group; Alternatively, subjects whose test values ​​are greater than the threshold have a greater risk of experiencing grade ≥ 2 immune-related adverse events after induced immunization than subjects whose test values ​​are less than or equal to the threshold.

6. Use of a marker in the preparation of a detection reagent, a detection kit or a device for predicting immune-related adverse events of locally advanced non-small cell lung cancer in the ICIs-CRT±ICIs treatment mode, characterized in that: The marker is CD8 + PD-1 + T.

7. The use according to claim 6, characterized in that: The detection reagents, detection kits or devices include anti-CD3-FITC-clone OKT3, anti-CD8-APC-clone RPA-T8, anti-CD279 (PD-1)-PE-clone EH12.2H7 and anti-IgG4Fc-clone HP6025.

8. The use according to claim 7, characterized in that: The applications include: Collect peripheral blood samples from individuals to be tested; Add flow cytometry antibodies to bind to peripheral blood T cell surface antigens; Use flow cytometry to detect T cell suspension and obtain CD8 + PD-1 + T in CD8 + The percentage value of T; and The risk and timing of individual immune-related adverse events ≥ grade 2 were predicted based on flow cytometer detection data and judgment thresholds.

9. The use according to claim 8, characterized in that: The individuals to be tested are patients with locally advanced non-small cell lung cancer under the ICIs-CRT±ICIs treatment model.

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