A biomarker for early diagnosis of non-small cell lung cancer and application thereof

Abstract

The application belongs to the technical field of biology and relates to a biomarker for early diagnosis of non-small cell lung cancer and application thereof. The biomarker comprises a combination of secreted phosphoprotein 1, epithelial cell adhesion molecule and matrix metallopeptidase 14, and / or a combination of a coding nucleic acid of secreted phosphoprotein 1, a coding nucleic acid of epithelial cell adhesion molecule and a coding nucleic acid of matrix metallopeptidase 14; the coding nucleic acid comprises mRNA or cDNA. A non-small cell lung cancer circulating tumor cell score diagnosis model constructed by taking the biomarker as a feature has a diagnosis accuracy of 87.5% in a test set, and the sensitivity and specificity are 92.5% and 82.5% respectively. Compared with existing tumor indexes in the clinic, the biomarker can detect cancer at an earlier stage and is conducive to early screening of non-small cell lung cancer.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a biomarker for the early diagnosis of non-small cell lung cancer and its application. Background Technology

[0002] Non-small cell lung cancer (NSCLC) is an important histological subtype of lung cancer. Despite numerous efforts and attempts over the years to achieve early diagnosis, most patients are still diagnosed at an intermediate or advanced stage. Traditional early diagnostic methods include imaging examinations, lung cancer cytology and histology, and serum tumor markers, but most have poor predictive sensitivity and specificity. Therefore, there is an urgent need to develop a highly sensitive, highly specific, and minimally invasive diagnostic method for NSCLC, which is of great significance for early detection of precancerous lesions, timely intervention, and improving patient survival rates.

[0003] Circulating tumor cells (CTCs) refer to tumor cells that detach from solid tumors and enter the peripheral blood circulation. They then travel to distant tissues, infiltrate, adapt to the new microenvironment, and eventually "seed," "proliferate," "colonize," and form metastatic lesions. They are the "seeds" of tumor metastasis. Therefore, CTCs, as a non-invasive diagnostic method of "liquid biopsy," have broad prospects.

[0004] Currently, many technologies have been applied to the isolation and enrichment of cytokines (CTCs), with epithelial cell adhesion molecules being one of the most commonly used biomarkers for isolating CTCs from lung cancer. However, primary lung cancer tumors exhibit significant heterogeneity, leading to the potential loss of mesenchymal phenotype CTCs by traditional CTC isolation methods that rely solely on epithelial cell adhesion molecules. Furthermore, numerous studies have confirmed that the expression profiles and contents of CTCs are highly similar to their parent cells, and the biomarkers they contain are closely related to cancer development and progression, aiding in diagnosis, providing deeper insights into their heterogeneity, and identifying drug resistance.

[0005] Therefore, there is an urgent need to develop a new early diagnostic biomarker and method for non-small cell lung cancer to further improve the accuracy of non-small cell lung cancer diagnosis. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a biomarker for the early diagnosis of non-small cell lung cancer and its application. Based on the biomarker, a model for the early diagnosis of non-small cell lung cancer is constructed, enabling accurate, efficient, and rapid diagnosis of early-stage non-small cell lung cancer.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a biomarker for the early diagnosis of non-small cell lung cancer, the biomarker comprising a combination of secretory phosphoprotein 1, epithelial cell adhesion molecule and matrix metallopeptidase 14, and / or a combination of nucleic acid encoding secretory phosphoprotein 1, nucleic acid encoding epithelial cell adhesion molecule and nucleic acid encoding matrix metallopeptidase 14; the encoded nucleic acid comprising mRNA or cDNA.

[0009] SEQ ID NO.1 (mRNA sequence of secreted phosphoprotein 1 NM_000582.3):

[0010]

[0011] SEQ ID NO.2 (mRNA sequence of epithelial cell adhesion molecule NM_002354.2):

[0012]

[0013] SEQ ID NO.3 (mRNA sequence of matrix metallopeptidase 14 NM_004995.3):

[0014]

[0015] The circulating tumor cell scoring diagnostic model for non-small cell lung cancer constructed using the biomarkers of this invention achieved a diagnostic accuracy of 87.5%, with sensitivity and specificity of 92.5% and 82.5%, respectively, in the training set. Compared to existing clinical tumor markers, it can detect cancer at an earlier stage, which is beneficial for the early screening of non-small cell lung cancer.

[0016] In a second aspect, the present invention provides the use of the biomarkers and / or detection reagents for the early diagnosis of non-small cell lung cancer as described in the first aspect in the preparation of products for the early diagnosis of non-small cell lung cancer.

[0017] Thirdly, the present invention provides a kit for the early diagnosis of non-small cell lung cancer, the kit comprising reagents for detecting the presence or expression level of the biomarkers for the early diagnosis of non-small cell lung cancer described in the first aspect.

[0018] Preferably, the reagent includes primers and / or probes for detecting the biomarkers for early diagnosis of non-small cell lung cancer as described in the first aspect.

[0019] Preferably, the kit further includes immunomagnetic beads modified with capture antibodies.

[0020] Preferably, the capturing antibody includes any one or a combination of at least two of the following: antibodies against epithelial adhesion molecules, antibodies against N-cadherin, or antibodies against epidermal growth factor.

[0021] This invention utilizes multi-antibody modified magnetic beads (EpCAM / N-cadherin / EGFR antibody) to target and capture circulating tumor cells (CTCs) specific to non-small cell lung cancer. The antibody can be mixed with the circulating tumor cell sample before or after mixing with the magnetic beads.

[0022] This invention can capture circulating tumor cells in peripheral blood from complex blood samples, effectively obtain biological information of NSCLC cancer sites for diagnosis, and non-destructively detect tumor status. The blood sample processing method is fast and simple, greatly simplifying the blood sample processing process before sample capture.

[0023] Fourthly, the present invention provides a method of using the reagent kit described in the third aspect, the method comprising the following steps:

[0024] (1) Using the capture antibody-modified immunomagnetic beads described in the third aspect to isolate circulating tumor cells of non-small cell lung cancer from biological samples;

[0025] (2) Extract RNA from circulating tumor cells of non-small cell lung cancer after lysis, or reverse transcribe it into cDNA;

[0026] (3) Quantitatively detect mRNA or cDNA using PCR and calculate the expression level of the mRNA of the biomarkers mentioned in the first aspect;

[0027] (4) Using the expression level of mRNA described in step (3) as a feature, construct an early diagnosis model for non-small cell lung cancer, obtain the score of circulating tumor cells of non-small cell lung cancer in the subject, and determine whether the subject is positive for non-small cell lung cancer based on the score.

[0028] Preferably, the early diagnostic model for non-small cell lung cancer includes a multivariate logistic regression model, wherein the output variable of the multivariate logistic regression model is the non-small cell lung cancer circulating tumor cell score of the subject, and the input variables of the multivariate logistic regression model are: the expression level of secretory phosphoprotein 1, the expression level of epithelial cell adhesion molecule, and the expression level of matrix metallopeptidase 14; or the expression level of nucleic acid encoded by secretory phosphoprotein 1, the expression level of nucleic acid encoded by epithelial cell adhesion molecule, and the expression level of nucleic acid encoded by matrix metallopeptidase 14.

[0029] Preferably, the formula for calculating the circulating tumor cell score of non-small cell lung cancer is as shown in equation (1):

[0030] NSCLC CTC Scores D =﹣9.311+0.480×SPP1+0.459×EPCAM+0.44×MMP14 (1);

[0031] Among them, NSCLC CTC Scores D The score represents the circulating tumor cells in non-small cell lung cancer. SPP1 represents the expression level of secreted phosphoprotein 1 or its encoded nucleic acid, EPCAM represents the expression level of epithelial cell adhesion molecule or its encoded nucleic acid, and MMP14 represents the expression level of matrix metallopeptidase 14 or its encoded nucleic acid.

[0032] Preferably, the criterion for judgment is: NSCLC CTC Scores D A value >-0.4554 indicates a positive result for non-small cell lung cancer.

[0033] Fifthly, the present invention provides a device for early diagnosis of non-small cell lung cancer, the device comprising an information acquisition module, a calculation module, and a diagnostic module;

[0034] The information acquisition module is used to perform the following:

[0035] Obtain the subject's detection information, which includes the expression level information of the biomarkers for early diagnosis of non-small cell lung cancer as described in the first aspect;

[0036] The calculation module is used to perform the following:

[0037] The expression level information of the obtained biomarkers was substituted into the early diagnosis model of non-small cell lung cancer to calculate the score of circulating tumor cells in non-small cell lung cancer. The calculation formula is shown in Equation (1):

[0038] NSCLC CTC Scores D =﹣9.311+0.480×SPP1+0.459×EPCAM+0.44×MMP14 (1);

[0039] Among them, NSCLC CTC Scores D The score represents the circulating tumor cells in non-small cell lung cancer. SPP1 represents the expression level of secreted phosphoprotein 1 or its encoded nucleic acid, EPCAM represents the expression level of epithelial cell adhesion molecule or its encoded nucleic acid, and MMP14 represents the expression level of matrix metallopeptidase 14 or its encoded nucleic acid.

[0040] The diagnostic module is used to perform the following:

[0041] The determination of whether a patient is positive for non-small cell lung cancer (NSCLC) is based on the calculated score of circulating tumor cells. The criteria for determination are: if the score of circulating tumor cells in NSCLC is > -0.4554, then the patient is considered positive for NSCLC.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention is the first to discover that secreted phosphoprotein 1, epithelial cell adhesion molecules, and matrix metallopeptidase 14 are closely related to the occurrence and development of NSCLC. The mRNA levels of these substances were detected, and a multivariate logistic regression model was used to establish an early diagnostic model for NSCLC. The diagnostic performance of this model is superior to that of the commonly used clinical serum tumor marker CEA. On the training set, the accuracy reaches 87.5%, with sensitivity and specificity of 92.5% and 82.5%, respectively. On the total sample, the accuracy reaches 89%, with sensitivity and specificity of 94% and 94%, respectively. This model demonstrates superior diagnostic performance for early-stage NSCLC and provides a methodological reference for the current early clinical diagnosis of NSCLC. Attached Figure Description

[0044] Figure 1 Flowchart for screening characteristic mRNA genes in NSCLC;

[0045] Figure 2A This is a graph showing the expression of characteristic genes related to the diagnostic model in normal and tumor tissues.

[0046] Figure 2B To train a heatmap of characteristic mRNA gene expression levels in peripheral blood PBMCs from benign pulmonary nodules;

[0047] Figure 2C A heatmap of characteristic mRNA gene expression levels in peripheral blood PBMCs of early non-small cell lung cancer for training set;

[0048] Figure 2D To validate the heatmap of characteristic mRNA gene expression levels in peripheral blood PBMCs from concentrated benign pulmonary nodules;

[0049] Figure 2E To validate the heatmap of characteristic mRNA gene expression levels in peripheral blood PBMCs from early-stage non-small cell lung cancer;

[0050] Figure 3 ROC curve of a single target gene in distinguishing between benign pulmonary nodules and early-stage non-small cell lung cancer patients;

[0051] Figure 4A A composite heatmap of the training set;

[0052] Figure 4B To train the CTC score of early-stage non-small cell lung cancer and benign pulmonary nodules D Result box plot;

[0053] Figure 4C ROC plots for early-stage non-small cell lung cancer and benign pulmonary nodules were used for training.

[0054] Figure 4D The diagnostic efficacy of the model was further evaluated using a confusion matrix algorithm;

[0055] Figure 5A To validate CTC scores in early-stage non-small cell lung cancer, benign pulmonary nodules, and other cancers. D Result box plot;

[0056] Figure 5B To validate the ROC plots for early-stage non-small cell lung cancer and other cancers;

[0057] Figure 5C CTC Scores for overall NSCLC and overall benign pulmonary nodules D ROC plot and serum tumor biomarker ROC plot;

[0058] Figure 5D CTC Scores for overall NSCLC and overall benign pulmonary nodules D Confusion matrix diagram. Detailed Implementation

[0059] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0060] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0061] In a specific embodiment of the present invention, NSCLC-derived circulating tumor cells (CTCs) in peripheral blood are targeted and captured based on multi-antibody modified magnetic beads (EpCAM / N-cadherin / EGFR antibody). The expression levels of characteristic mRNAs in circulating tumor cells are detected by droplet digital PCR (ddPCR) technology to determine whether the subject has NSCLC. Furthermore, the CTC ScoreD diagnostic model established by multivariate logistic regression and the expression level of serum carcinoembryonic antigen (CEA) are used to determine whether cancer is present. The diagnostic performance is evaluated using ROC plots and confusion matrix plots.

[0062] In one specific embodiment of the present invention, a kit for early diagnosis of non-small cell lung cancer is provided. The kit includes reagents for detecting the presence or expression level of biomarkers for early diagnosis of non-small cell lung cancer as described in the present invention, such as SPP1 probe (Hs00959010_m1), CEACAM5 probe (Hs00944025_m1), EPCAM probe (Hs00901885_m1), KRT5 probe (Hs00361185_m1), KRT19 probe (Hs01051611_gH), MMP14 probe (Hs00237119_m1) and immunomagnetic beads modified with capture antibodies. The capture antibodies include any one or a combination of at least two of the following: antibodies against epithelial adhesion molecules, antibodies against N-cadherin, or antibodies against epidermal growth factor.

[0063] In one specific embodiment of the present invention, a method for using a reagent kit for the early diagnosis of non-small cell lung cancer is provided, the method comprising the following steps:

[0064] (1) Isolating circulating tumor cells of non-small cell lung cancer from biological samples using immunomagnetic beads modified with capture antibodies;

[0065] (2) RNA was extracted from circulating tumor cells of non-small cell lung cancer after lysis and reverse transcribed into cDNA;

[0066] (3) Quantitative detection of cDNA was performed using PCR, and the expression levels of the mRNA corresponding to the cDNA of the biomarkers secretory phosphoprotein 1, epithelial cell adhesion molecule and matrix metallopeptidase 14 described in this invention were calculated.

[0067] (4) Using the expression level of mRNA described in step (3) as a feature, construct an early diagnosis model for non-small cell lung cancer, obtain the score of circulating tumor cells of non-small cell lung cancer in the subject, and determine whether the subject is positive for non-small cell lung cancer based on the score.

[0068] The non-small cell lung cancer early diagnosis model includes a multivariate logistic regression model. The output variable of the multivariate logistic regression model is the non-small cell lung cancer circulating tumor cell score of the subject. The input variables of the multivariate logistic regression model are: the expression level of secretory phosphoprotein 1, the expression level of epithelial cell adhesion molecule, and the expression level of matrix metallopeptidase 14; or the expression level of nucleic acid encoded by secretory phosphoprotein 1, the expression level of nucleic acid encoded by epithelial cell adhesion molecule, and the expression level of nucleic acid encoded by matrix metallopeptidase 14.

[0069] The formula for calculating the circulating tumor cell score in non-small cell lung cancer is shown in equation (1):

[0070] NSCLC CTC Scores D =﹣9.311+0.480×SPP1+0.459×EPCAM+0.44×MMP14 Equation (1).

[0071] Among them, NSCLC CTC Scores D The score represents the circulating tumor cells in non-small cell lung cancer. SPP1 represents the expression level of secreted phosphoprotein 1 or its encoded nucleic acid, EPCAM represents the expression level of epithelial cell adhesion molecule or its encoded nucleic acid, and MMP14 represents the expression level of matrix metallopeptidase 14 or its encoded nucleic acid.

[0072] The criteria for judgment are: NSCLC CTC Scores D A value >-0.4554 indicates a positive result for non-small cell lung cancer.

[0073] In one specific embodiment of the present invention, a device for early diagnosis of non-small cell lung cancer is provided, the device comprising an information acquisition module, a calculation module and a diagnostic module.

[0074] The information acquisition module is used to perform the following:

[0075] Obtain the subject's detection information, which includes the expression level information of the biomarker for early diagnosis of non-small cell lung cancer as described in claim 1.

[0076] The calculation module is used to perform the following:

[0077] The expression level information of the obtained biomarkers was substituted into the early diagnosis model of non-small cell lung cancer to calculate the score of circulating tumor cells in non-small cell lung cancer. The calculation formula is shown in Equation (1):

[0078] NSCLC CTC Scores D =﹣9.311+0.480×SPP1+0.459×EPCAM+0.44×MMP14 (1);

[0079] Among them, NSCLC CTC Scores D The score represents the circulating tumor cells in non-small cell lung cancer. SPP1 represents the expression level of secreted phosphoprotein 1 or its encoded nucleic acid, EPCAM represents the expression level of epithelial cell adhesion molecule or its encoded nucleic acid, and MMP14 represents the expression level of matrix metallopeptidase 14 or its encoded nucleic acid.

[0080] The diagnostic module is used to perform the following:

[0081] The determination of whether a patient is positive for non-small cell lung cancer (NSCLC) is based on the calculated score of circulating tumor cells. The criteria for determination are: if the score of circulating tumor cells in NSCLC is > -0.4554, then the patient is considered positive for NSCLC. Example

[0082] This embodiment describes the construction and evaluation of an early diagnostic model based on the characteristic mRNA of NSCLC CTCs.

[0083] A total of 112 participants were recruited, including patients with benign pulmonary nodules, early-stage NSCLC, and other cancers. All participants were enrolled in the study between February 2024 and December 2024. Among them were 50 patients with stage I-II non-small cell lung cancer, 50 patients with benign pulmonary nodules, and 12 patients with other cancers. Peripheral blood samples were collected from a total of 112 participants.

[0084] The inclusion criteria for the study participants were as follows: 1) Patients who had not yet received surgery, radiotherapy, chemotherapy, or other anti-tumor treatments at the time of initial diagnosis; 2) Patients without hematological diseases; 3) Patients who understood and agreed to participate in this study and had signed the informed consent form. The exclusion criteria were as follows: 1) Patients with a history of other malignant tumors; 2) Patients with severe infections or heart, lung, liver, or kidney dysfunction; 3) Patients who experienced insufficient blood volume, coagulation, or hemolysis after blood sample collection. In accordance with relevant medical ethics review methods, all steps and procedures of this experiment were approved by the Ethics Review Committee of the Second Affiliated Hospital of Soochow University (Approval No. #JD-LK2024021-I01), and all participants provided informed consent for this study.

[0085] (1) Screening of characteristic mRNA genes in NSCLC

[0086] Screening process as follows Figure 1 As shown, firstly, using the adenocarcinoma datasets GSE19188, GSE75037, GSE27262, and GSE63459, and the squamous cell carcinoma datasets GSE19188, GSE2088, and GSE51852 from the Gene Expression Omnibus (GEO) dataset, differentially expressed genes that are overexpressed in NSCLC tissues but lowly expressed or not expressed in normal lung tissues were screened as candidate genes using the Limma package in R. The screening criteria were set as: logFC ≥ 1 and P < 0.05. Then, based on the HPA immune cell dataset within the Human Protein Atlas (HPA), candidate genes were further screened to exclude genes highly expressed in immune cells. Ultimately, six target gene mRNAs specifically highly expressed in NSCLC were identified: secreted phosphoprotein 1 (SPP1), carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), epithelial cell adhesion molecule (EPCAM), keratin 5 (KRT5), keratin 19 (KRT19), and matrix metallopeptidase 14 (MMP14). Simultaneously, the expression of these six genes in lung and NSCLC tissues was queried in the TCGA and GTEx databases. Figure 2A The results showed that these six genes could distinguish between tumors and normal tissues, with SPP1, EPCAM, MMP14, and KRT19 being expressed at higher levels in cancerous tissues.

[0087] (2) Isolation of CTCs and RNA extraction from subject blood samples

[0088] Four mL of peripheral venous blood was collected from each subject in an EDTA vacuum anticoagulation blood collection tube. After sampling, the tube was inverted five times and treated at 4°C for use within 24 hours. To avoid epithelial cell contamination, all blood samples were from peripheral blood outside the first tube. The blood collection tube was centrifuged at 300 g for 15 min in a horizontal centrifuge at room temperature to obtain plasma and the lower layer of blood cells. After aspirating the upper plasma, 1×PBS solution was added to the blood cell pellet to dilute it to 10 mL (twice the initial peripheral blood volume), and the mixture was thoroughly mixed. SepMate was then used to collect the final sample. TMPeripheral blood mononuclear cell (PBMC) layers were separated in centrifuge tubes, washed, and then resuspended in 400 μL (0.1 times the initial peripheral blood volume) of 1×PBS solution. 200 μL of the PBMC suspension (corresponding to 2 mL of initial peripheral blood volume) was incubated with 0.1 mg of biotinylated multi-antibody (EpCAM / N-caherin / EGFR, antibody purchase information is detailed in Table 1) modified magnetic beads at 25°C for 30 min to capture non-small cell lung cancer (CTC) cells. The remaining PBMC suspension was added to 700 μL of QIAzol lysis buffer and stored at -20°C for later use. After washing the CTC samples twice, 700 μL of QIAzol lysis buffer was added, and RNA was extracted using the Direct-zol™ RNA Microprep kit (ZYMO RESEARCH) and reverse transcribed into cDNA template using a reverse transcription kit (Takara). The cDNA template was then stored at -20°C for later use.

[0089] Table 1

[0090]

[0091] (3) Quantification of characteristic mRNA genes of CTCs using ddPCR

[0092] Based on the target gene mRNA coding gene sequence screened in step (1), target gene primers and probes (Thermo Fisher) were ordered. The target gene mRNA molecules in the samples were detected using a QX200Auto DG Droplet Digital PCR (ddPCR) system to obtain the mRNA expression level of the target gene in NSCLC CTCs captured in each sample (2 mL corresponding to peripheral blood samples). The ddPCR reaction system volume was 20 μL, consisting of ddPCR probe premix (dUTP-free), enzyme-free sterile water, cDNA template, target gene primers and probes. After amplifying the target gene cDNA template by PCR, the cDNA corresponding to the target gene mRNAs in the sample (transcripts / μL) was quantitatively detected using a QX200 Droplet Reader. The mRNA expression level of the target gene in NSCLC CTCs captured in each 2 mL peripheral blood sample was calculated. A training set of 40 patients with benign pulmonary nodules and 40 patients with early-stage NSCLC was used, while a validation set of 10 patients with benign pulmonary nodules, 10 patients with early-stage NSCLC, and 12 patients with other cancers was used. The results of the training set are shown below. Figure 2B , Figure 2C As shown, the validation set detection results are as follows: Figure 2D , Figure 2E As shown.

[0093] Nucleic acid detection probes are commercially available products, as shown in Table 2.

[0094] Table 2

[0095]

[0096] (4) Construction of an early diagnosis model for non-small cell lung cancer and evaluation of its diagnostic efficacy

[0097] Based on the mRNA expression levels in preoperative blood samples from NSCLC patients and blood samples from patients with benign pulmonary nodules obtained in step (3), a diagnosis was performed using the device designed in this invention for early diagnosis of non-small cell lung cancer. The results were analyzed using ROC analysis to calculate the area under the curve (AUC) and to evaluate the diagnostic efficacy of a single target gene. Figure 3 Genes were selected as modeling features based on their AUC value (>0.70) and p value (p<0.05), namely SPP1, EPCAM, and MMP14.

[0098] A diagnostic model for NSCLC CTC RNA was established using SPSS 27.0 software through multivariate logistic regression analysis. NSCLC CTC Scores D = -9.311+0.480×SPP1+0.459×EPCAM+0.44×MMP14, by substituting the expression levels of the modeling feature genes in the sample into the model, the box plot and composite heatmap corresponding to the NSCLC CTC Scores for each subject sample can be obtained. Figure 4A , Figure 4B ).

[0099] The diagnostic effectiveness of the NSCLC diagnostic model was evaluated using ROC curves. The results showed that, under optimal conditions, the model achieved a sensitivity and specificity of 92.5% and 82.5% respectively for diagnosing patients in the training set, with a diagnostic accuracy of 87.5% and an AUC of 0.91. Figures 4C-4D In a small validation set, results showed significant differences between other cancers and benign lung nodules and early-stage NSCLC. Figure 5A The AUC value for other cancers and early-stage NSCLC reached 0.98 ( Figure 5B In summary, in the overall sample (including 50 cases of NSCLC, 50 cases of benign pulmonary nodules, and 12 cases of other cancers), the diagnostic sensitivity and specificity were 94% and 84%, respectively, and the diagnostic accuracy reached 89%. Figure 5D To compare the diagnostic capabilities of NSCLC diagnostic models with traditional non-small cell lung cancer tumor markers such as CEA, the AUCs for modeling and diagnosis using CEA, CA199, and CA125 were 0.7, 0.73, and 0.73, respectively, with clinically acceptable cutoff values. Figure 5C Therefore, it can be seen that the NSCLC early diagnosis model established in this invention has better diagnostic performance for non-small cell lung cancer than commonly used clinical serum tumor markers.

[0100] In summary, the circulating tumor cell scoring diagnostic model for non-small cell lung cancer, constructed using the biomarkers of this invention, achieved a diagnostic accuracy of 87.5%, with sensitivity and specificity of 92.5% and 82.5%, respectively, in the test set. Compared to existing clinical tumor markers, it can detect cancer at an earlier stage, which is beneficial for the early screening of non-small cell lung cancer.

[0101] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. The application of a reagent for detecting the presence or expression level of biomarkers used in the early diagnosis of non-small cell lung cancer in the preparation of a kit for the early diagnosis of non-small cell lung cancer, characterized in that, The kit includes reagents for detecting the presence or expression level of biomarkers for early diagnosis of non-small cell lung cancer and immunomagnetic beads modified with capture antibodies; the capture antibodies include a combination of antibodies against epithelial adhesion molecules, antibodies against N-cadherin, and antibodies against epidermal growth factor. The method of using the kit includes the following steps: (1) Using the immunomagnetic beads modified with the capture antibody to isolate circulating tumor cells of non-small cell lung cancer from biological samples; (2) Extract RNA from circulating tumor cells of non-small cell lung cancer after lysis, or reverse transcribe it into cDNA; (3) Quantitative detection of mRNA or cDNA using PCR, and calculation of the expression level of mRNA for biomarkers used in the early diagnosis of non-small cell lung cancer; (4) Using the expression level of mRNA described in step (3) as a feature, construct an early diagnosis model for non-small cell lung cancer, obtain the score of circulating tumor cells of non-small cell lung cancer in the subject, and determine whether the subject is positive for non-small cell lung cancer based on the score; The early diagnostic model for non-small cell lung cancer includes a multivariate logistic regression model. The output variable of the multivariate logistic regression model is the non-small cell lung cancer circulating tumor cell score of the subject. The input variables of the multivariate logistic regression model are: the expression level of secretory phosphoprotein 1 encoded nucleic acid, the expression level of epithelial cell adhesion molecule encoded nucleic acid, and the expression level of matrix metallopeptidase 14 encoded nucleic acid. The formula for calculating the circulating tumor cell score in non-small cell lung cancer is shown in equation (1): NSCLC CTC Scores D =﹣9.311+0.480×SPP1+0.459×EPCAM+0.44×MMP14 (1); Among them, NSCLC CTC Scores D The score represents the circulating tumor cells in non-small cell lung cancer. SPP1 represents the expression level of nucleic acid encoded by secreted phosphoprotein 1, EPCAM represents the expression level of nucleic acid encoded by epithelial cell adhesion molecule, and MMP14 represents the expression level of nucleic acid encoded by matrix metallopeptidase 14. The criteria for judgment are: NSCLC CTC Scores D A value >-0.4554 indicates a positive result for non-small cell lung cancer; The biomarkers for the early diagnosis of non-small cell lung cancer include a combination of nucleic acids encoding secretory phosphoprotein 1, nucleic acids encoding epithelial cell adhesion molecule, and nucleic acids encoding matrix metallopeptidase 14; the encoded nucleic acids include mRNA or cDNA.

2. The application of the reagent according to claim 1 for detecting the presence or expression level of biomarkers for early diagnosis of non-small cell lung cancer in the preparation of a kit for early diagnosis of non-small cell lung cancer, characterized in that, The reagents include primers and / or probes for biomarkers used in the early diagnosis of non-small cell lung cancer.

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