Application of plasma extracellular vesicle miRNA marker in preparation of product for predicting curative effect of lung cancer immune combined chemotherapy

Abstract

The invention provides an application of a plasma extracellular vesicle miRNA (micro Ribonucleic Acid) marker in preparation of a product for predicting the curative effect of lung cancer immune combined chemotherapy. The miRNA marker comprises any one or a combination of at least two of miR-18a-3p, miR-23a-3p, miR-29a-3p, miR-181d-5p, miR-330-3p or miR-4508. The expression level of miRNA of plasma extracellular vesicles of lung cancer patients is detected by adopting a small RNA sequencing technology, the patients are grouped according to an imaging evaluation result after immune combined chemotherapy, and miRNA biomarkers which can be used for precisely subdividing and monitoring the curative effects of beneficial people after lung cancer immune combined chemotherapy are explored by using a statistical method. The miRNA biomarker has high sensitivity and convenience, and has an important value for clinical application scene subdivision of an immune combined chemotherapy treatment scheme.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically relates to the application of plasma extracellular vesicle miRNA markers in the preparation of a product for predicting the efficacy of combined immunotherapy and chemotherapy for lung cancer. Background Art

[0002] Lung cancer is the most common primary malignant tumor of the lung. Over the past 50 years, the incidence and mortality of lung cancer have increased rapidly, ranking first among the causes of cancer death in the world. At present, conventional methods for treating lung cancer include surgical treatment, platinum-containing doublet chemotherapy, radiotherapy, targeted therapy and immunotherapy. In recent years, immunotherapy combined with chemotherapy has become an important interventional therapy for lung cancer. This combination regimen can significantly improve the efficacy without the superposition of obvious drug-related adverse reactions. It is mainly suitable for the maintenance treatment of advanced lung cancer and neoadjuvant and adjuvant treatment of locally advanced lung cancer. Its high effectiveness and good safety have gradually become the main treatment mode for advanced lung cancer. If the potential beneficiary population can be reasonably screened and the efficacy of the beneficiary population can be accurately subdivided before the treatment is implemented, it will provide important information for individualized precision treatment.

[0003] Currently, the main biomarkers for screening patients potentially benefiting from immunotherapy include PD-L1 expression, tumor mutational burden (TMB), and microsatellite instability (MSI). High MSI (MSI-high, MSI-H) is a screening marker for immune checkpoint inhibitors in colorectal cancer, but it is extremely rare in lung cancer patients, making it difficult to predict immune-related efficacy in lung cancer patients. PD-L1 expression on tumor cells may influence the efficacy of immune checkpoint inhibitors in lung cancer patients, but heterogeneity in this assay is a significant issue. Different antibodies, different detection platforms, and different assessment systems have different positive cutoffs, making it difficult to establish a consistent standard for measuring PD-L1 expression on tumor cells. Furthermore, its accuracy and specificity are low. Even in patients with the highest PD-L1 expression (≥50%), the response rate is only 45.2%. TMB has been shown to correlate with immunotherapy efficacy in multiple large-scale clinical trials and can complement PD-L1 testing. However, TMB prediction currently suffers from limited specificity and limitations, requiring further prospective studies. While more and more patients are benefiting from immunotherapy combined with chemotherapy, effective biomarkers for accurately screening potential beneficiaries and precisely segmenting the efficacy of these patients remain lacking. Exploring predictive biomarkers, accurately screening responders to tumor immunotherapy combined with chemotherapy, and precisely segmenting and monitoring the efficacy of these patients are crucial to reducing unnecessary waste of medical resources, enabling timely adjustments to treatment strategies, and improving the efficacy of immunotherapy combined with chemotherapy.

[0004] Small extracellular vesicles (sEVs) are vesicles with a lipid bilayer membrane structure secreted by cells, typically 30-150 nm in diameter. The contents of sEVs, such as lipids, proteins, and nucleic acids, play a crucial role in intercellular communication. Extracellular vesicles secreted by different cells have different content compositions and functions. These differences can be exploited to use sEVs and their contents as biomarkers for disease diagnosis. Small RNAs in sEVs, including microRNAs (miRNAs), have been considered potential biomarkers for various cancers. However, it is unclear whether sEV miRNAs can serve as biomarkers to predict the efficacy of combined immunotherapy and chemotherapy and to accurately segment and monitor the efficacy of the benefiting population.

[0005] Furthermore, widely used clinical imaging tests (such as chest enhanced CT, chest X-ray, MRI (Magnetic Resonance Imaging, MRI), and ultrasound as auxiliary imaging tests) and tumor marker tests (such as CEA (carcinoembryonic antigen, carcinoembryonic antigen), NSE (neuron-specific enolase, neuron-specific enolase, CYFRA21-1, proGRP, and SCC-Ag (squamous cell carcinoma antigen)) have low sensitivity and specificity for lung cancer efficacy detection and are prone to false positives. There are also no reports on highly specific blood extracellular vesicle miRNA biomarkers for predicting the efficacy of combined immunotherapy and chemotherapy in patients with lung adenocarcinoma, especially for accurately segmenting and monitoring the efficacy of the benefiting population.

[0006] Currently, immunotherapy combined with chemotherapy has become an important option for the clinical treatment of lung cancer patients, and an increasing number of patients have benefited from immunotherapy combined with chemotherapy. However, biomarkers that effectively identify true responders are still lacking. Therefore, developing a method based on sEVs miRNA markers that can effectively indicate and monitor the efficacy of immunotherapy combined with chemotherapy in lung cancer patients will help clinicians conduct accurate individualized assessments of patients with advanced lung adenocarcinoma, thereby bringing better survival benefits to patients and facilitating more precise clinical application of immunotherapy combined with chemotherapy. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention aims to provide the use of plasma extracellular vesicle miRNA markers in the preparation of a product for predicting the efficacy of combined immunotherapy and chemotherapy for lung cancer. Based on the expression levels of miRNA biomarkers derived from plasma extracellular vesicles in patients with lung adenocarcinoma, the present invention establishes a prognostic assessment kit for combined immunotherapy and chemotherapy in lung cancer patients. This kit can effectively indicate and monitor the efficacy of combined immunotherapy and chemotherapy in lung cancer patients, helping clinicians to conduct accurate individualized assessments of patients with advanced lung adenocarcinoma, thereby bringing better survival benefits to patients. It is of great value in the subdivision of clinical application scenarios of combined immunotherapy and chemotherapy treatment regimens.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides the use of plasma extracellular vesicle miRNA markers in the preparation of a product for predicting the efficacy of combined immunotherapy and chemotherapy for lung cancer, wherein the plasma extracellular vesicle miRNA markers include: any one of miR-18a-3p, miR-23a-3p, miR-29a-3p, miR-181d-5p, miR-330-3p or miR-4508, or a combination of at least two of them.

[0010] Through analysis and screening of plasma extracellular vesicle miRNA expression profiles and treatment effects in patients with advanced lung adenocarcinoma before treatment, the present invention discovered plasma exosome-derived miRNAs biomarkers that can be used to predict the efficacy of combined immunotherapy and chemotherapy, especially to accurately segment and monitor the efficacy of the beneficiary population. These biomarkers can effectively indicate the efficacy of combined immunotherapy and chemotherapy in lung cancer patients, help clinicians conduct accurate individualized assessments of patients with advanced lung adenocarcinoma, and thus bring better survival benefits to patients.

[0011] The present invention adopts the plasma extracellular vesicle small RNA sequencing strategy and discovers for the first time 6 lung cancer plasma extracellular vesicle miRNAs: miR-18a-3p, miR-23a-3p, miR-29a-3p, miR-181d-5p, miR-330-3p and miR-4508, which can be used as biomarkers for monitoring the efficacy of immunotherapy combined with chemotherapy treatment for lung cancer with high sensitivity and convenience.

[0012] Preferably, the lung cancer immunotherapy combined with chemotherapy efficacy prediction product predicts that the subject is a responder to lung cancer immunotherapy combined with chemotherapy, or predicts that the subject is a non-responder to lung cancer immunotherapy combined with chemotherapy based on the relative expression level of the plasma extracellular vesicle miRNA marker.

[0013] Preferably, compared with the disease stable group, the subject whose expression level of any one or a combination of at least two of the miR-23a-3p, miR-29a-3p, miR-181d-5p or miR-330-3p is upregulated, or the subject whose expression level of miR-18a-3p and / or miR-4508 is downregulated is a responder to combined immunotherapy and chemotherapy for lung cancer.

[0014] In the present invention, whether it is up-regulated miRNA or down-regulated miRNA, as long as the expression level of one or more miRNAs undergoes the expected change.

[0015] Preferably, the immunotherapy in the lung cancer combined with chemotherapy includes PD-1 inhibitor therapy, and the chemotherapy includes pemetrexed and / or platinum chemotherapy.

[0016] Preferably, the PD-1 inhibitor is selected from pembrolizumab.

[0017] Preferably, the platinum is selected from carboplatin.

[0018] In the present invention, the regimen of lung cancer immunotherapy combined with chemotherapy is pembrolizumab (K drug, PD-1 inhibitor) + pemetrexed + carboplatin.

[0019] The present invention found that compared with the stable disease group, lung cancer patients with upregulated expression levels of any one or a combination of at least two of the following extracellular vesicle miR-23a-3p, miR-29a-3p, miR-181d-5p, or miR-330-3p in plasma experienced partial disease remission after immunotherapy combined with chemotherapy and may benefit from the pembrolizumab combined chemotherapy treatment regimen. Compared with the stable disease group, lung cancer patients with downregulated expression levels of miR-18a-3p or miR-4508 in plasma extracellular vesicles experienced partial disease remission after immunotherapy combined with chemotherapy and may benefit from the pembrolizumab combined chemotherapy treatment regimen.

[0020] In a second aspect, the present invention provides the use of a detection reagent for the expression level of plasma extracellular vesicle miRNA markers in the preparation of a product for predicting the efficacy of lung cancer combined with immunotherapy and chemotherapy, wherein the detection reagent includes: primers and probes for detecting any one or a combination of at least two of miR-18a-3p, miR-23a-3p, miR-29a-3p, miR-181d-5p, miR-330-3p or miR-4508.

[0021] Preferably, the immunotherapy in the lung cancer combined with chemotherapy includes PD-1 inhibitor therapy, and the chemotherapy includes pemetrexed and / or platinum chemotherapy.

[0022] Preferably, the PD-1 inhibitor is selected from pembrolizumab.

[0023] Preferably, the platinum is selected from carboplatin.

[0024] In a third aspect, the present invention provides a kit for predicting the efficacy of combined immunotherapy and chemotherapy for lung cancer, which comprises primers and probes for detecting any one or a combination of at least two of miR-18a-3p, miR-23a-3p, miR-29a-3p, miR-181d-5p, miR-330-3p or miR-4508.

[0025] Preferably, the kit further comprises: any one or a combination of at least two of a reagent for extracting plasma extracellular vesicles, a reagent for extracting plasma small extracellular vesicle miRNA, or a PCR detection reagent.

[0026] In the present invention, extracellular vesicle (exosome) separation reagent L3525 (3Dmed, Shanghai) is used to extract plasma extracellular vesicles. In addition, plasma extracellular vesicle extraction methods also include: centrifugation (differential centrifugation, density gradient centrifugation), particle size separation methods (ultrafiltration, exclusion chromatography), immunoaffinity, microfluidics technology and other commercial exosome isolation kits.

[0027] In a fourth aspect, the present invention provides a model for predicting the efficacy of combined immunotherapy and chemotherapy for lung cancer, the model comprising:

[0028] A data acquisition unit: obtaining the relative expression level of plasma extracellular vesicle miRNA markers of the subject before lung cancer immunotherapy combined with chemotherapy, wherein the plasma extracellular vesicle miRNA markers include: any one of miR-18a-3p, miR-23a-3p, miR-29a-3p, miR-181d-5p, miR-330-3p or miR-4508, or a combination of at least two thereof;

[0029] Efficacy prediction unit: Based on the relative expression levels of the plasma extracellular vesicle miRNA markers, judging whether the subject is a responder to lung cancer immunotherapy combined with chemotherapy; the judgment criteria include: compared with the disease stable group, the subject whose expression level of any one or a combination of at least two of the miR-23a-3p, miR-29a-3p, miR-181d-5p or miR-330-3p is upregulated, or the subject whose expression level of miR-18a-3p and / or miR-4508 is downregulated is a responder to lung cancer immunotherapy combined with chemotherapy.

[0030] In the present invention, small RNA sequencing technology is used to detect the expression level of plasma extracellular vesicle miRNA. In addition, qRT-PCR, miRNA chip detection and third-generation sequencing technology can also be used to detect the expression level of plasma extracellular vesicle miRNA.

[0031] In the present invention, a biomarker model is established using one of the contents of blood extracellular vesicles, miRNA, to identify people who benefit from the immunotherapy combined with chemotherapy treatment for lung cancer. In addition, there are many other contents of extracellular vesicles, such as lncRNA, circRNA, mRNA, piRNA, proteins, lipids and small molecule metabolites. These blood extracellular vesicle contents may also construct a biomarker model to achieve similar effects as miRNA. In addition, various components in the patient's blood, such as extracellular free nucleic acids (cfRNA and cfDNA), CTCs (Circulating tumor cells), ctDNA (Circulating tumor DNA) and protein biomarkers can be used as biomarkers to predict the efficacy of immunotherapy combined with chemotherapy for lung cancer.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] At present, the imaging examinations and tumor marker tests widely used in clinical practice have low sensitivity and specificity for detecting the efficacy of lung cancer, and cannot be directly used to predict the efficacy of combined immunotherapy and chemotherapy. There are no research reports on highly specific blood extracellular vesicle miRNA biomarkers for predicting the efficacy of combined immunotherapy and chemotherapy in patients with lung adenocarcinoma and for accurately segmenting and monitoring the efficacy of the beneficiary population.

[0034] Studies on the prediction of the efficacy of immunotherapy combined with chemotherapy in patients with lung adenocarcinoma have the following characteristics: a. The number of studies focusing on the prediction of the efficacy of immunotherapy combined with chemotherapy in patients with lung adenocarcinoma is relatively small; b. The number of studies based on the second-generation sequencing platform is relatively small; c. There are relatively few studies on extracellular vesicle contents as biomarkers for predicting the efficacy of immunotherapy combined with chemotherapy in patients with lung adenocarcinoma; d. There are no research reports proposing extracellular vesicle miRNA as a highly specific biomarker to screen for patients who will benefit from immunotherapy combined with chemotherapy and to accurately segment and monitor the efficacy of these patients.

[0035] This invention analyzes and screens the plasma extracellular vesicle miRNA expression profiles and treatment effects of patients with advanced lung adenocarcinoma before treatment, and discovers plasma exosome-derived miRNAs biomarkers that can be used to predict the efficacy of combined immunotherapy and chemotherapy and screen potential beneficiaries. These biomarkers can effectively indicate the efficacy of combined immunotherapy and chemotherapy in lung cancer patients, help clinicians conduct accurate individualized assessments of patients with advanced lung adenocarcinoma, and thus bring better survival benefits to patients. They are of great value for the segmentation of clinical application scenarios of combined immunotherapy and chemotherapy treatment regimens.

[0036] Specifically, the present invention uses a plasma extracellular vesicle small RNA sequencing strategy to discover for the first time six lung cancer plasma extracellular vesicle miRNAs: miR-18a-3p, miR-23a-3p, miR-29a-3p, miR-181d-5p, miR-330-3p and miR-4508, which can be used as biomarkers for monitoring the efficacy of lung cancer immunotherapy combined with chemotherapy, with high sensitivity and convenience. The present invention found that compared with the disease stable group, lung cancer patients with upregulated expression levels of any one or a combination of at least two of plasma extracellular vesicle miR-23a-3p, miR-29a-3p, miR-181d-5p or miR-330-3p, or lung cancer patients with downregulated expression levels of plasma extracellular vesicle miR-18a-3p and / or miR-4508, experienced partial remission of the disease after immunotherapy combined with chemotherapy and could benefit from the pembrolizumab combined with chemotherapy treatment regimen.

[0037] The present invention adopts liquid biopsy identification technology, which can be combined with imaging examinations to improve the specificity of monitoring the efficacy of the population responding to the immunotherapy combined with chemotherapy treatment regimen, reduce false positives, and avoid overmedicalization. It can be used to accurately screen the potential population that will benefit from the immunotherapy combined with chemotherapy for lung cancer, especially to accurately segment and monitor the efficacy of the beneficiary population. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the TEM detection result of plasma extracellular vesicles.

[0039] Figure 2 This is the result of plasma extracellular vesicle NTA detection.

[0040] Figure 3 This is a graph showing the expression of plasma extracellular vesicle miR-18a-3p between the partial remission group and the stable disease group.

[0041] Figure 4 This is a graph showing the expression of plasma extracellular vesicle miR-23a-3p between the partial remission group and the stable disease group.

[0042] Figure 5 This is a graph showing the expression of plasma extracellular vesicle miR-29a-3p between the partial remission group and the stable disease group.

[0043] Figure 6 This is a graph showing the expression of plasma extracellular vesicle miR-181d-5p between the partial remission group and the stable disease group.

[0044] Figure 7 This is a graph showing the expression of plasma extracellular vesicle miR-330-3p between the partial remission group and the stable disease group.

[0045] Figure 8This is a graph showing the expression of plasma extracellular vesicle miR-4508 between the partial remission group and the stable disease group. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0047] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0048] Example 1

[0049] 1. Study cohort and clinical information

[0050] This study enrolled 25 patients with advanced or metastatic lung adenocarcinoma. Blood samples were collected from each patient before drug treatment for plasma extracellular vesicle extraction and subsequent analysis. Patient inclusion criteria were as follows:

[0051] 1) Pathological diagnosis of lung adenocarcinoma.

[0052] 2) Patients receiving the "pembrolizumab (K drug, PD-1 inhibitor) + pemetrexed + carboplatin" treatment regimen for the first time and have no history of chemotherapy.

[0053] 3) There are efficacy evaluation data.

[0054] One month after the drug treatment, the efficacy was evaluated according to the RECIST1.1 standard for the evaluation of efficacy in solid tumors. 14 patients had partial response (PR) and 11 patients had stable disease (SD).

[0055] 2. Plasma Extracellular Vesicle Extraction and Characterization

[0056] 2.1 Plasma collection and extracellular vesicle extraction

[0057] Blood samples were collected from patients before they began immunotherapy and chemotherapy. Blood samples were stored in 10 mL vacutainer tubes (REF367525, BD, USA), gently inverted several times, and then placed upright. Samples were centrifuged at 1600 g for 10 minutes at 4°C. Samples with hemolysis grade less than 4 after centrifugation were used for subsequent studies. The supernatant was transferred to a 1.5 mL EP tube and centrifuged at 16,000 g for 15 minutes at 4°C to remove residual cell debris. The supernatant was then aliquoted into 1.5 mL EP tubes and stored at −80°C until use.

[0058] Frozen plasma samples were thawed in a 37°C water bath and centrifuged at 12,000 g for 10 min at 4°C. The supernatant was filtered sequentially through a 0.45 μm filter column (CLS8163-100EA, Corning, USA) and then a 0.22 μm filter column (CLS8161-100EA, Corning, USA). The supernatant was centrifuged at 12,000 g for 5 min at 4°C and the filtrate was collected into a 2 mL EP tube. The volume of the filtrate was measured, and 1 / 4 volume of exosome precipitation reagent L3525 (3Dmed, Shanghai) was added. The tube was mixed by vortexing and incubated at 4°C for 30 min. The supernatant was discarded and the extracellular vesicles were resuspended in 200 μL of PBS.

[0059] 2.2 Characterization of plasma extracellular vesicles

[0060] TEM detection: Extracellular vesicles were resuspended in PBS and fixed with 4% paraformaldehyde. The fixed extracellular vesicles were dropped onto carbon-coated copper grids. After incubation at room temperature for 5 minutes, the cells were rinsed twice with PBS, rinsed once with PBS containing glycine (50mM), and incubated with PBS containing 0.5% BSA for 10 minutes. Finally, the copper grids were negatively stained with 2% uranyl acetate for 10 minutes and observed and photographed using a transmission electron microscope (H-7650, Hitachi, Japan). The morphology of extracellular vesicles was detected by TEM, and the results showed that the extracellular vesicles showed a typical "horseshoe-shaped" morphology (see Figure 1 ).

[0061] NTA assay: First, plasma extracellular vesicles were diluted with PBS to 1×10 7 -1×10 9 / mL, pipette and mix. Subsequently, turn on the NTA instrument (NanoSight NS300, Malvern, UK) and inject the sample into the sample chamber. Use the 488nm excitation module, set the camera lens parameters, select the shutter value of 890, the gain value of 146, and the detection threshold of 7. At least 200 complete tracks were analyzed and obtained for each video. Finally, the nanoparticle tracking data of extracellular vesicles were analyzed using NTA version 2.3 analysis software. The particle size distribution of extracellular vesicles was detected by NTA, and the results showed that the average particle size of the extracellular vesicles was 114.9nm, which is consistent with the particle size distribution of extracellular vesicles (see Figure 2 ).

[0062] 3. Molecular marker discovery

[0063] 3.1 Extraction of plasma extracellular vesicle miRNA

[0064] Total RNA was extracted from extracellular vesicles using the miRNeasy Serum / Plasma Kit (217184, QIAGEN, Shanghai) according to the product instructions. RNA was eluted with 15 μL of nuclease-free water. The concentration and fragment distribution of miRNAs were determined using an Agilent 2100 Bioanalyzer and the accompanying small RNA analysis kit (5067-1548, Agilent).

[0065] 3.2 Construction of plasma extracellular vesicle miRNA library

[0066] miRNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (E7300L, NEB, USA) according to the product instructions. Six μL of each RNA sample was loaded, followed by 18 cycles of 3'-end adapter ligation, reverse transcription primer hybridization, 5'-end adapter ligation, reverse transcription, and PCR amplification. PCR amplification products were purified using the NucleoSpinGel and PCR Clean-up kit (740609.250, MN, Germany), and the library DNA was eluted with 30 μL of elution buffer. The library DNA concentration was determined using an Invitrogen Qubit 4 fluorometer and the accompanying reagent Qubit dsDNA HS Assay Kit (Q32854, Thermofisher, USA). The distribution of library DNA fragments was determined using an Agilent 2100 bioanalyzer and the accompanying chip and reagents Agilent High Sensitivity DNA Kit & Reagents (5067-4626, Agilent, USA). The sequencing was performed using the Illumina NovaSeq platform with a 2x150 bp paired-end sequencing strategy, and the sequencing data volume of each library was 6G.

[0067] 3.3 Sequencing data analysis process

[0068] Using small RNA sequencing technology, we obtained the expression levels of extracellular vesicle miRNAs in the plasma of lung cancer patients. The analysis process of the sequencing data is as follows:

[0069] Sequencing data alignment: After removing the sequencing adapters of the small RNA sequencing detection data, the sequencing data were aligned to the human reference genome hg19 (genome download link: http: / / hgdownload.soe.ucsc.edu / goldenPath / hg19 / bigZips / ) using BWA software (version: 0.7.12-r1039), and the number of reads aligned to the miRNA was counted.

[0070] miRNA annotation: miRNAs were annotated using the Gencode v25 and miRBase v21 databases, and those annotated as known mature miRNAs were retained for subsequent analysis.

[0071] miRNA filtering: Mature miRNAs with a length of 30 nt or less and covered by at least 2 reads per sample were retained for subsequent analysis.

[0072] Normalization of miRNA expression: The trimmed mean of M-values (TMM) method of the edgeR v3.26.8 analysis package in R language and the voom method of the limma v3.40.6 analysis package were used to normalize the miRNA expression of the samples.

[0073] Based on the expression of miRNA in plasma extracellular vesicles of lung cancer patients, samples were grouped according to imaging evaluation results after immunotherapy combined with chemotherapy. Statistical methods were used to discover plasma extracellular vesicle miRNAs as biomarkers that can be used to accurately segment and monitor the efficacy of immunotherapy combined with chemotherapy in lung cancer patients. The process is as follows:

[0074] Sample grouping: The samples were grouped according to the imaging evaluation results after the patients' immunotherapy combined with chemotherapy, and divided into two groups: partial remission group (PR) and stable disease group (SD).

[0075] Candidate molecular markers: The linear model fitting (limma-voom) method in the limma analysis package of R language was used to analyze the miRNAs with significantly different expression levels between the PR group and the SD group. MiRNAs with an expression level CPM (Counts Per Million) greater than 32, a change of more than 1.5 times between the two groups, and a test result of P < 0.05 were selected as candidate molecular markers.

[0076] Using statistical methods to discover miRNA biomarkers that can be used to accurately segment and monitor the efficacy of lung cancer immunotherapy combined with chemotherapy in the beneficiary population: miR-18a-3p ( Figure 3 )、miR-23a-3p( Figure 4 )、miR-29a-3p( Figure 5 )、miR-181d-5p( Figure 6 )、miR-330-3p( Figure 7 ) and miR-4508( Figure 8 ) A total of 6 types.

[0077] miR-18a-3p (SEQ ID NO: 1): ACUGCCCUAAAGUGCUCCUUCUGG.

[0078] miR-23a-3p (SEQ ID NO:2): AUCACAUUGCCAGGGAUUUCC.

[0079] miR-29a-3p (SEQ ID NO:3): UAGCACCAUCUGAAAUCGGUUA.

[0080] miR-181d-5p (SEQ ID NO:4): AACAUUCAUUGUUGUCGGUGGGU.

[0081] miR-330-3p (SEQ ID NO:5): GCAAAGCACACGGCCUGCAGAGA.

[0082] miR-4508 (SEQ ID NO:6): GCGGGGCUGGGCGCCG.

[0083] Lung cancer patients with high plasma extracellular vesicle expression levels of miR-23a-3p, miR-29a-3p, miR-181d-5p, or miR-330-3p who achieved partial disease remission after immunotherapy combined with chemotherapy could benefit from pembrolizumab combined with chemotherapy. Conversely, patients with low expression levels of these four miRNAs experienced stable disease after immunotherapy combined with chemotherapy and had less benefit from pembrolizumab combined with chemotherapy. Lung cancer patients with low plasma extracellular vesicle expression levels of miR-18a-3p or miR-4508 experienced partial disease remission after immunotherapy combined with chemotherapy and could benefit from pembrolizumab combined with chemotherapy. Conversely, patients with high expression levels of these two miRNAs experienced stable disease after immunotherapy combined with chemotherapy and had less benefit from pembrolizumab combined with chemotherapy.

[0084] In summary, the present invention discovered plasma exosome-derived miRNAs biomarkers that can be used to predict the efficacy of combined immunotherapy and chemotherapy and screen potential beneficiaries through analysis and screening of the plasma extracellular vesicle miRNA expression profiles and treatment effects of patients with advanced lung adenocarcinoma before treatment. These biomarkers can effectively indicate the efficacy of combined immunotherapy and chemotherapy in lung cancer patients, help clinicians conduct accurate individualized assessments of patients with advanced lung adenocarcinoma, thereby bringing better survival benefits to patients, and are of great value for further subdividing the clinical application scenarios of combined immunotherapy and chemotherapy treatment regimens.

[0085] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. Application of plasma extracellular vesicle miRNA markers in the preparation of a product for predicting the efficacy of combined immunotherapy and chemotherapy for lung cancer, characterized in that: The plasma extracellular vesicle miRNA marker includes: any one of miR-18a-3p, miR-23a-3p, miR-29a-3p, miR-181d-5p, miR-330-3p or miR-4508, or a combination of at least two thereof.

2. The use according to claim 1, characterized in that The lung cancer immunotherapy combined with chemotherapy efficacy prediction product predicts that a subject is a responder to lung cancer immunotherapy combined with chemotherapy, or predicts that a subject is a non-responder to lung cancer immunotherapy combined with chemotherapy based on the relative expression level of the plasma extracellular vesicle miRNA marker.

3. The use according to claim 2, characterized in that Compared with the disease stable group, the subjects whose expression levels of any one or a combination of at least two of the miR-23a-3p, miR-29a-3p, miR-181d-5p or miR-330-3p are upregulated, or whose expression levels of miR-18a-3p and / or miR-4508 are downregulated are responders to combined immunotherapy and chemotherapy for lung cancer.

4. The use according to claim 3, characterized in that The immunotherapy in the lung cancer combined with chemotherapy includes PD-1 inhibitor therapy, and the chemotherapy includes pemetrexed and / or platinum chemotherapy; The PD-1 inhibitor is selected from pembrolizumab; The platinum is selected from carboplatin.

5. The use of a detection reagent for the expression level of plasma extracellular vesicle miRNA markers in the preparation of a product for predicting the efficacy of combined immunotherapy and chemotherapy for lung cancer, characterized in that: The detection reagent includes primers and probes for detecting any one or a combination of at least two of miR-18a-3p, miR-23a-3p, miR-29a-3p, miR-181d-5p, miR-330-3p or miR-4508.

6. The use according to claim 5, characterized in that The immunotherapy in the lung cancer combined immunotherapy and chemotherapy includes PD-1 inhibitor therapy, and the chemotherapy includes pemetrexed and / or platinum chemotherapy.

7. The use according to claim 6, characterized in that The PD-1 inhibitor is selected from pembrolizumab; The platinum is selected from carboplatin.

8. A kit for predicting the efficacy of combined immunotherapy and chemotherapy for lung cancer, characterized in that: The kit includes primers and probes for detecting any one or a combination of at least two of miR-18a-3p, miR-23a-3p, miR-29a-3p, miR-181d-5p, miR-330-3p or miR-4508.

9. The kit according to claim 8, characterized in that The kit further comprises: any one or a combination of at least two of a reagent for extracting plasma extracellular vesicles, a reagent for extracting plasma small extracellular vesicle miRNA, or a PCR detection reagent.

10. A model for predicting the efficacy of combined immunotherapy and chemotherapy for lung cancer, characterized in that: The model includes: A data acquisition unit: obtaining the relative expression level of plasma extracellular vesicle miRNA markers of the subject before lung cancer immunotherapy combined with chemotherapy, wherein the plasma extracellular vesicle miRNA markers include: any one of miR-18a-3p, miR-23a-3p, miR-29a-3p, miR-181d-5p, miR-330-3p or miR-4508, or a combination of at least two thereof; Efficacy prediction unit: Based on the relative expression levels of the plasma extracellular vesicle miRNA markers, judging whether the subject is a responder to lung cancer immunotherapy combined with chemotherapy; the judgment criteria include: compared with the disease stable group, the subject whose expression level of any one or a combination of at least two of the miR-23a-3p, miR-29a-3p, miR-181d-5p or miR-330-3p is upregulated, or the subject whose expression level of miR-18a-3p and / or miR-4508 is downregulated is a responder to lung cancer immunotherapy combined with chemotherapy.

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