A marker for predicting the curative effect and prognosis of NSCLC immunotherapy and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to RAB8A as a biomarker for predicting the curative effect and prognosis of immunotherapy of non-small cell lung cancer (NSCLC) patients and application thereof. Through independent discovery and verification queues, it is proved that high expression of RAB8A in tumor tissues is significantly related to longer progression-free survival of NSCLC patients after receiving immunotherapy, and the expression level has good prediction efficiency on the curative effect and prognosis of immunotherapy. Detection of RAB8A protein expression can be used as an effective auxiliary tool for guiding the clinical decision of NSCLC immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to RAB8A as a biomarker for predicting the efficacy and prognosis of immunotherapy in patients with non-small cell lung cancer (NSCLC) and its application. Background Technology

[0002] Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for more than 85% of all lung cancer cases. In recent years, the application of immune checkpoint inhibitors (ICIs) has greatly improved the treatment landscape for advanced NSCLC, significantly prolonging patients' overall survival (OS) and progression-free survival (PFS).

[0003] However, immunotherapy still faces significant challenges in practice: only a subset of patients benefit from it, and more than half face primary or secondary resistance. Currently, widely used predictive biomarkers, such as the expression level of programmed death-ligand-1 (PD-L1), have limited predictive efficacy and a large number of false positives and false negatives. Emerging biomarkers such as tumor mutational burden (TMB) are limited in clinical application due to high testing costs and a lack of standardized protocols.

[0004] Therefore, finding and identifying novel biomarkers that can more accurately and conveniently predict the efficacy of NSCLC immunotherapy is of great clinical significance and application value for achieving personalized precision treatment and avoiding the toxic side effects and economic burden caused by ineffective treatment. Summary of the Invention

[0005] The purpose of this invention is to provide a biomarker for predicting the efficacy and prognosis of immunotherapy for NSCLC and its application. Through immunohistochemistry, a significant correlation was found between the expression level of RAB8A protein in tumor tissue and the progression-free survival (PFS) of NSCLC patients after receiving immunotherapy, providing a new solution to the above problems.

[0006] To achieve the above objectives, this application employs the following technical solution:

[0007] A biomarker for predicting the efficacy and prognosis of immunotherapy for NSCLC, the biomarker being the RAB8A protein in tumor tissue.

[0008] Furthermore, the biomarker functions by detecting the expression level of RAB8A protein in the subject's tumor tissue.

[0009] Furthermore, when the RAB8A protein expression level in the subject's tumor tissue was higher than the optimal cutoff value, the subject responded better to immunotherapy and had a longer progression-free survival.

[0010] Furthermore, the optimal cutoff value was determined through receiver operating characteristic (ROC) curve analysis.

[0011] Furthermore, the area under the ROC curve of the predictive performance was greater than the set threshold.

[0012] Furthermore, it also includes using tumor tissue RAB8A protein in combination with tissue PD-L1 as a biomarker.

[0013] Application of a biomarker for predicting the efficacy and prognosis of NSCLC immunotherapy, wherein the biomarker for predicting the efficacy and prognosis of NSCLC immunotherapy is used in the preparation of reagents or kits for predicting the efficacy of NSCLC immunotherapy.

[0014] The beneficial effects of this invention are:

[0015] This invention reveals a positive correlation between RAB8A protein expression levels in NSCLC tumor tissue and the efficacy and prognosis of immunotherapy. RAB8A, as a novel biomarker, offers the following advantages:

[0016] 1. High predictive accuracy: In both the discovery and validation cohorts, patients with high RAB8A expression were shown to have significantly longer progression-free survival, with the area under the curve (AUC) of predictive efficacy greater than 0.7, demonstrating good discriminative ability.

[0017] 2. Intuitive results and mature technology: Immunohistochemical detection methods have been widely used in clinical pathology departments. The technology is mature and stable, and the results are easy to interpret, making it easy to promote on existing clinical platforms.

[0018] 3. Great potential for clinical application: This invention provides new and reliable molecular markers for screening NSCLC patients who may benefit from immunotherapy, which helps guide clinical treatment decisions, avoids the use of expensive immunotherapies for ineffective patients, thereby achieving personalized precision treatment, improving patient benefits and saving medical resources. Attached Figure Description

[0019] Figure 1 Representative immunohistochemical results revealing low levels of RAB8A expression in tumor tissues of patients in the cohort.

[0020] Figure 2 Representative immunohistochemical results revealing high levels of RAB8A expression in tumor tissues of patients in the cohort.

[0021] Figure 3The study compared the expression levels of RAB8A in tumor tissues of patients with long and short progression-free survival (PFS) in the cohort.

[0022] Figure 4 The study compared progression-free survival (PFS) between patients with high and low RAB8A expression in the cohort.

[0023] Figure 5 The ROC curves of RAB8A expression levels in the cohort predicting the prognosis of immunotherapy were discovered.

[0024] Figure 6 The ROC curves of tissue PD-L1 expression levels in the cohort were found to predict the prognosis of immunotherapy.

[0025] Figure 7 The ROC curves of RAB8A combined with tissue PD-L1 in the cohort were found to predict the prognosis of immunotherapy.

[0026] Figure 8 . Validate representative immunohistochemical results of low-level RAB8A expression in tumor tissues of patients in the validation cohort.

[0027] Figure 9 . Validate representative immunohistochemical results of high-level RAB8A expression in tumor tissues of patients in the cohort.

[0028] Figure 10 Comparison of RAB8A expression levels in tumor tissues of patients with long and short PFS in the validation cohort.

[0029] Figure 11 Comparison of progression-free survival (PFS) between patients with high and low RAB8A expression in the validation cohort.

[0030] Figure 12 . Validate the ROC curve of RAB8A expression level in the cohort to predict the prognosis of immunotherapy.

[0031] Figure 13 . Validate the ROC curves of tissue PD-L1 expression levels in the cohort to predict the prognosis of immunotherapy.

[0032] Figure 14 ROC curves for predicting immunotherapy prognosis in the cohort using RAB8A combined with tissue PD-L1. Detailed Implementation

[0033] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0034] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0035] Example 1: Patient Cohort and Samples

[0036] 1. Subject screening

[0037] This retrospective study included patients in the hospital's oncology department who were pathologically diagnosed with advanced NSCLC and were receiving immune checkpoint inhibitors (single-agent or in combination with chemotherapy) as first-line or later-line treatment.

[0038] 2. Selection Criteria

[0039] Age ≥18 years; histologically or cytologically diagnosed NSCLC; having at least one measurable lesion; having received at least 2 cycles of immunotherapy; having complete follow-up imaging data and survival data.

[0040] 3. Exclusion criteria

[0041] Patients with incomplete clinical pathology data; patients lost to follow-up during the follow-up period; and patients with other active malignant tumors.

[0042] 4. Patient grouping

[0043] Progression-free survival (PFS) was used as the primary efficacy endpoint. A total of 64 patients (31 with squamous cell carcinoma and 33 with adenocarcinoma) were randomly assigned using stratified random sampling:

[0044] Discovery cohort: 31 patients, used for preliminary exploration and determination of the predictive cutoff value for RAB8A.

[0045] Validation cohort: 33 patients, used to independently validate the predictive power of the above cutoff values.

[0046] Example 2 reveals immunohistochemical (IHC) detection of RAB8A protein expression in the cohort.

[0047] A. Test Methods

[0048] 1. Continuously cut 4μm thick sections from the paraffin-embedded tumor tissue of the patient before treatment.

[0049] 2. After dewaxing and hydration, the sections were subjected to heat-induced antigen retrieval using EDTA antigen retrieval solution (pH 9.0).

[0050] 3. After eliminating endogenous peroxidase, add anti-RAB8A rabbit monoclonal antibody (primary antibody) and incubate overnight at 4°C.

[0051] 4. The next day, add HRP-labeled secondary antibody and incubate at room temperature for 30 minutes.

[0052] 5. Develop the stain using DAB chromogenic agent, counterstain the cell nuclei with hematoxylin, and mount with neutral resin.

[0053] 6. Two senior pathologists, unfamiliar with the clinical pathology data and treatment efficacy information, independently reviewed the slides. The H-Score was calculated based on a combination of staining intensity and the percentage of positive cells.

[0054] B. Test Results

[0055] IHC analysis successfully detected RAB8A protein expression in tumor tissues from 31 NSCLC patients, with expression localized in the cytoplasm. Low RAB8A expression was observed, specifically occasional brown staining in the tumor cell cytoplasm. Figure 1 RAB8A is highly expressed, meaning that there is a relatively strong brown staining in the cytoplasm of most tumor cells. Figure 2 Typical IHC staining results are as follows: Figure 1 , 2 As shown.

[0056] Example 3 revealed an association between RAB8A expression in the cohort and progression-free survival (PFS) in patients.

[0057] A. Test Methods

[0058] Progression-free survival (PFS) data were collected from all patients via telephone follow-up or by reviewing the electronic medical record system. PFS was defined as the time from the start of immunotherapy to disease progression or death from any cause. Survival curves were plotted using the Kaplan-Meier method, and intergroup comparisons were performed using the Log-rank test.

[0059] B. Test Results

[0060] Based on the median progression-free survival (PFS), all patients were divided into two groups: long PFS and short PFS. Patients with long PFS showed significantly higher RAB8A expression levels than those in the short PFS group. Figure 3 H-Score = 8 vs. H-Score = 6, P = 0.0019. Based on a RAB8A expression cutoff value of 6.0, all patients were divided into a high-expression group and a low-expression group. The median PFS in the high-expression group was significantly longer than that in the low-expression group. Figure 4 (24.1 months vs. 5.5 months, P = 0.0002).

[0061] Example 4: Evaluation and Comparison of Predictive Performance of RAB8A in the Discovery Cohort

[0062] A. Test Methods

[0063] Using the patient's PFS status (whether the disease progresses within 6 months) after receiving immunotherapy as the standard, receiver operating characteristic (ROC) curves were plotted to predict efficacy based on RAB8A expression level (H-Score).

[0064] B. Test Results

[0065] In the discovery queue ( Figure 5 The ROC curve of RAB8A in predicting patient PFS showed a sensitivity of 75.00% (95% CI: 50.50%-89.82%), a specificity of 73.33% (95% CI: 48.05%-89.10%), and an AUC of 0.8063. Figure 5 The study found that the sensitivity of PD-L1 prediction in cohort organization was 76.92% (95% CI: 49.74%-91.82%), the specificity was 60.00% (95% CI: 35.75%-80.18%), and the AUC was 0.6769. Figure 6 ).

[0066] The above results indicate that tumor tissue RAB8A is a predictor of good prognosis and efficacy of immunotherapy in NSCLC patients, and its predictive efficacy is superior to that of tissue PD-L1.

[0067] Example 6: Evaluation of the predictive efficacy of cohort RAB8A in coordinating PD-L1

[0068] In the discovery cohort, a joint model of RAB8A and PD-L1 was established, with a predictive sensitivity of 92.31% (95% CI: 66.69%-99.61%), a specificity of 66.67% (95% CI: 41.71%-84.82%), and an AUC of 0.8256, indicating that the predictive power of the joint model is higher than that of any single indicator.

[0069] Example 7: Immunohistochemical (IHC) detection of RAB8A protein expression in the validation cohort

[0070] A. Test Methods

[0071] Same test method as in Example 2.

[0072] B. Test Results

[0073] IHC analysis successfully detected RAB8A protein expression in tumor tissues from 33 NSCLC patients, with expression localized in the cytoplasm. Similarly, RAB8A expression was low, only occasionally observed as brown staining in the tumor cell cytoplasm. Figure 8 RAB8A is highly expressed, meaning that there is a relatively strong brown staining in the cytoplasm of most tumor cells. Figure 9Typical IHC staining results are as follows: Figure 8 , 9 As shown.

[0074] Example 8: Association analysis of RAB8A expression and progression-free survival (PFS) in the validation cohort.

[0075] A. Test Methods

[0076] The method is the same as that in Example 3.

[0077] B. Test Results

[0078] Based on the median progression-free survival (PFS), all patients were divided into two groups: long PFS and short PFS. Patients with long PFS showed significantly higher RAB8A expression levels than those in the short PFS group. Figure 10 H-Score = 9 vs. H-Score = 4, P = 0.0076. Based on a RAB8A expression cutoff value of 6.0, all patients were divided into a high-expression group and a low-expression group. The median PFS in the high-expression group was significantly longer than that in the low-expression group. Figure 11 (15.6 months vs. 5.2 months, P = 0.0356).

[0079] Example 9: Evaluation and Comparison of Predictive Performance of the RAB8A Validation Cohort

[0080] A. Test Methods

[0081] Using the patient's PFS status (whether the disease progresses within 6 months) after receiving immunotherapy as the standard, receiver operating characteristic (ROC) curves were plotted to predict efficacy based on RAB8A expression level (H-Score).

[0082] B. Test Results

[0083] In the validation cohort, the ROC curve of RAB8A in predicting patient PFS showed a sensitivity of 75.00% (95% CI: 50.50%-89.82%), a specificity of 70.59% (95% CI: 46.87%-86.72%), and an AUC of 0.7647. Figure 12 The sensitivity of the validation cohort organization for PD-L1 prediction was 64.29% (95% CI: 38.76%-83.66%), the specificity was 78.57% (95% CI: 52.41%-92.43%), and the AUC was 0.6199. Figure 13 ).

[0084] The above results further demonstrate that tumor tissue RAB8A is a predictor of good prognosis and efficacy of immunotherapy in NSCLC patients, and its predictive efficacy is superior to that of tissue PD-L1.

[0085] Example 10: Joint analysis of the predictive power of the RAB8A cohort and clinical standard PD-L1.

[0086] In the validation cohort, a joint model of RAB8A and PD-L1 was established, with a sensitivity of 85.71% (95% CI: 60.06%-97.46%), a specificity of 64.29% (95% CI: 38.76% - 83.66%), and an AUC of 0.7781, indicating that the predictive power of the joint model is higher than that of any single indicator.

[0087] This invention, through independent discovery and validation cohort studies, demonstrates that high expression of RAB8A in tumor tissue is significantly associated with longer progression-free survival in NSCLC patients after immunotherapy, and that its expression level has good predictive power for immunotherapy efficacy and prognosis. Detecting RAB8A protein expression can serve as an effective auxiliary tool to guide clinical decision-making in NSCLC immunotherapy. The combination of RAB8A and tissue PD-L1 can further enhance predictive efficacy.

[0088] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting the expression level of RAB8A protein in tumor tissue in the preparation of reagents or kits for predicting the efficacy of immunotherapy for NSCLC.

2. The application according to claim 1, characterized in that, When the RAB8A protein expression level in a subject's tumor tissue is higher than the optimal cutoff value, the subject responds better to immunotherapy and has a longer progression-free survival.

3. The application according to claim 2, characterized in that, The optimal cutoff value was determined through receiver operating characteristic (ROC) curve analysis.

4. The application according to claim 3, characterized in that, The area under the ROC curve of the predictive performance was greater than the set threshold.

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