Serum protein biomarker for lung cancer typing and application thereof

Abstract

The invention discloses a serum protein biomarker for lung cancer typing and application of the serum protein biomarker. The serum protein biomarker for lung cancer typing comprises an APOC2 protein and / or an APOC4 protein. The biomarker disclosed by the invention can be used for effectively distinguishing lung cancer types, and the APOC2 protein and the APOC4 protein have relatively high diagnosis efficiency (AUC is 0.72 and 0.80 respectively) on NSCLC and SCLC in a discovery set. In an external verification queue, the optimal thresholds of APOC2 and APOC4 are determined to be 9.226 ng / mL and 5.094 ng / mL respectively, good diagnostic performance (AUC is 0.99 and 0.88 respectively) is shown, and the reliability and practicability of APOC2 and APOC4 in precise lung cancer typing are indicated.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a serum protein biomarker for lung cancer typing and its application. Background Art

[0002] Lung cancer is mainly divided into two pathological subtypes: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), and there are significant differences in molecular mechanisms, treatment responses, and prognoses between the two. NSCLC is usually treated with surgical resection, targeted therapy, and immunotherapy, while the breakthrough of immunotherapy for SCLC is limited, and there are few effective targets, mainly relying on chemotherapy and radiotherapy. Compared with NSCLC, SCLC has a worse prognosis and a higher recurrence rate. After NSCLC patients develop resistance to targeted therapy, the treatment plan can be adjusted through genetic testing, while 80% of SCLC patients relapse within one year after treatment, often accompanied by brain metastasis. Therefore, achieving accurate diagnosis of NSCLC and SCLC is of great significance for the diagnosis and treatment of lung cancer: on the one hand, it can avoid the abuse of targeted drugs caused by misdiagnosing SCLC as NSCLC; on the other hand, it can prevent misjudging resectable NSCLC as SCLC and avoid a cliff-like decline in the 5-year survival rate of patients.

[0003] Currently, the differentiation between NSCLC and SCLC highly depends on histopathological examination. However, as an invasive examination method, tissue biopsy has the following limitations: biopsy operations may bring invasive risks and potential complications; the heterogeneity of tumor tissues may affect the accuracy of test results; and the test results of tissue biopsy have certain timeliness deficiencies. In contrast, serum protein biomarkers, as a non-invasive detection method, can be obtained through routine blood tests, avoiding the risks of invasive biopsies. In addition, some proteins are abnormally expressed in the early stage of tumorigenesis and have the potential to dynamically detect tumor progression. With the advantages of non-invasiveness, dynamic monitoring ability, and multi-dimensional information integration, serum protein biomarkers have become a core part of the tumor precision diagnosis and treatment system. Therefore, screening serum protein biomarkers that can be used for the accurate diagnosis of NSCLC and SCLC is expected to achieve the precise typing and personalized medicine of lung cancer, thereby improving the overall prevention and treatment level of lung cancer.

[0004] Existing biomarkers such as neuron-specific enolase (NSE) and progastrin releasing peptide (ProGRP) generally have an area under the Receiver Operating Characteristic Curve (ROC) value of less than 0.85 when diagnosing NSCLC and SCLC, which cannot meet the requirements of precise clinical typing of lung cancer.

[0005] Therefore, there is an urgent need to provide a specific biomarker and method that can effectively distinguish malignant tumors from inflammatory diseases. Summary of the Invention

[0006] In view of the deficiencies of the prior art and the actual needs, the present invention provides a serum protein biomarker for lung cancer typing and its application, which is screened based on ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). Specifically, the present invention performs proteomic analysis on the serum samples of NSCLC and SCLC patients, screens out the proteins with significant differences between the two groups, and uses enzyme-linked immunosorbent assay (ELISA) to verify in an independent cohort, determines the optimal diagnostic thresholds of APOC2 and APOC4 proteins, and finds that they exhibit excellent diagnostic performance and can be used as biomarkers for precise lung cancer typing.

[0007] To achieve the object of the present invention, the following technical solutions are adopted:

[0008] In a first aspect, the present invention provides a serum protein biomarker for lung cancer typing, and the serum protein biomarker for lung cancer typing includes: APOC2 protein and / or APOC4 protein.

[0009] The biomarker APOC2 protein and / or APOC4 protein of the present invention can effectively classify lung cancer. In the discovery set, the AUC value of APOC2 protein is 0.72, and the AUC value of APOC4 protein is 0.80. In the external validation cohort, the optimal diagnostic thresholds of APOC2 protein and APOC4 protein are determined to be 9.226 and 5.094 ng / mL respectively. The diagnostic accuracy of APOC2 is 96%, the sensitivity is 100%, the specificity is 93%, and the AUC value is 0.99; the diagnostic accuracy of APOC4 protein is 86%, the sensitivity is 71%, the specificity is 100%, and the AUC value is 0.88.

[0010] Preferably, the lung cancer includes non-small cell lung cancer and / or small cell lung cancer.

[0011] In a second aspect, the present invention provides the use of the serum protein biomarker and / or its detection reagent for lung cancer typing described in the first aspect in the preparation of a product for lung cancer typing.

[0012] In a third aspect, the present invention provides a kit for lung cancer typing, and the kit includes a reagent for detecting the presence or expression level of the serum protein biomarker for lung cancer typing described in the first aspect.

[0013] In a fourth aspect, the present invention provides a device for lung cancer typing, and the device includes a detection unit and an analysis unit;

[0014] The detection unit is used to perform including: detecting the expression level of APOC2 protein and / or APOC4 protein in a sample to be tested;

[0015] The analysis unit is used to perform including: judging whether the patient is a non-small cell lung cancer patient or a small cell lung cancer patient according to the expression level of APOC2 protein and / or APOC4 protein in the sample to be tested.

[0016] Preferably, the sample to be tested includes serum, preferably the serum of a lung cancer patient.

[0017] Preferably, the judgment criteria are:

[0018] (1) When the concentration of APOC2 protein < 9.226 ng / mL, it is judged as positive for non-small cell lung cancer, and when the concentration of APOC2 protein ≥ 9.226 ng / mL, it is judged as positive for small cell lung cancer; or,

[0019] (2) When the concentration of APOC4 protein < 5.094 ng / mL, it is judged as positive for non-small cell lung cancer, and when the concentration of APOC4 protein ≥ 5.094 ng / mL, it is judged as positive for small cell lung cancer.

[0020] Fifth aspect, the present invention provides a model for lung cancer typing, wherein the input variables of the model for lung cancer typing are the concentrations of APOC2 protein or APOC4 protein, and the output variables are positive for non-small cell lung cancer or positive for small cell lung cancer.

[0021] Preferably, the lung cancer typing includes non-small cell lung cancer and small cell lung cancer.

[0022] Preferably, the judgment criteria for the output variables are as follows:

[0023] (1) When the concentration of APOC2 protein < 9.226 ng / mL, it is judged as positive for non-small cell lung cancer; when the concentration of APOC2 protein ≥ 9.226 ng / mL, it is judged as positive for small cell lung cancer; or,

[0024] (2) When the concentration of APOC4 protein < 5.094 ng / mL, it is judged as positive for non-small cell lung cancer; when the concentration of APOC4 protein ≥ 5.094 ng / mL, it is judged as positive for small cell lung cancer.

[0025] Sixth aspect, the present invention provides the application of the serum protein biomarker for lung cancer typing described in the first aspect, the device for lung cancer typing described in the fourth aspect, or the model for lung cancer typing described in the fifth aspect in screening drugs for treating non-small cell lung cancer and / or small cell lung cancer.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention discovers for the first time the biomarker APOC2 protein and APOC4 protein for lung cancer typing, which can achieve accurate typing of lung cancer. The two proteins have high sensitivity and specificity in distinguishing NSCLC from SCLC, superior to the biomarkers with an AUC value generally lower than 0.85 in the prior art;

[0028] (2) The biomarker APOC2 protein and APOC4 protein of the present invention are significantly up-regulated in SCLC patients, and both show good diagnostic performance in the discovery set and the external validation cohort. In the discovery set, the AUC value of APOC2 protein is 0.72, and the AUC value of APOC4 protein is 0.80. In the external validation cohort, the optimal diagnostic thresholds of APOC2 protein and APOC4 protein are determined to be 9.226 and 5.094 ng / mL respectively. The diagnostic accuracy of APOC2 is 96%, the sensitivity is 100%, the specificity is 93%, and the AUC value is 0.99; the diagnostic accuracy of APOC4 protein is 86%, the sensitivity is 71%, the specificity is 100%, and the AUC value is 0.88. Description of the Drawings

[0029] Figure 1The differential box plot of 7 differential proteins in the discovery set;

[0030] Figure 2 The differential box plot of 7 differential proteins in the external validation set;

[0031] Figure 3 The ROC curve graph of APOC2 and APOC4 proteins for differentiating NSCLC from SCLC. Detailed implementation manners

[0032] To further elaborate on the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with embodiments and drawings. It can be understood that the specific implementation manners described herein are only used to explain the present invention, rather than limiting the present invention.

[0033] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For those reagents or instruments not specifying the manufacturer, they are all conventional products that can be commercially purchased through regular channels.

[0034] In a specific embodiment of the present invention, a kit for lung cancer typing is provided. The kit includes reagents for detecting the presence or expression level of the serum protein biomarkers for lung cancer typing described in the present invention, such as chemiluminescent immunoassay reagents (including APOC2 / APOC4 specific monoclonal antibody pairs), enzyme-linked immunosorbent assay (ELISA) kits (including APOC2 / APOC4 capture antibodies and HRP-labeled detection antibodies), or protein chip detection reagents (coated with APOC2 / APOC4 specific probes), any one or a combination of at least two of them.

[0035] In a specific embodiment of the present invention, a device for lung cancer typing is provided. The device includes a detection unit and an analysis unit;

[0036] The detection unit is used to perform the following: detecting the expression levels of APOC2 protein and / or APOC4 protein in a sample to be tested;

[0037] The analysis unit is used to perform the following: judging whether the patient is a non-small cell lung cancer patient or a small cell lung cancer patient according to the expression levels of APOC2 protein and / or APOC4 protein in the sample to be tested.

[0038] The sample to be tested includes serum, preferably the serum of a lung cancer patient.

[0039] The criteria for judgment are as follows:

[0040] (1) When the concentration of APOC2 protein < 9.226 ng / mL, it is judged as positive for non-small cell lung cancer; when the concentration of APOC2 protein ≥ 9.226 ng / mL, it is judged as positive for small cell lung cancer; or,

[0041] (2) When the concentration of APOC4 protein < 5.094 ng / mL, it is judged as positive for non-small cell lung cancer; when the concentration of APOC4 protein ≥ 5.094 ng / mL, it is judged as positive for small cell lung cancer.

[0042] In a specific embodiment of the present invention, a model for lung cancer typing is provided. The input variable of the model for lung cancer typing is the concentration of APOC2 protein or the concentration of APOC4 protein, and the output variable is positive for non-small cell lung cancer or positive for small cell lung cancer.

[0043] The lung cancer typing includes non-small cell lung cancer and / or small cell lung cancer.

[0044] The criteria for judgment are as follows:

[0045] (1) When the concentration of APOC2 protein < 9.226 ng / mL, it is judged as positive for non-small cell lung cancer; when the concentration of APOC2 protein ≥ 9.226 ng / mL, it is judged as positive for small cell lung cancer; or,

[0046] (2) When the concentration of APOC4 protein < 5.094 ng / mL, it is judged as positive for non-small cell lung cancer; when the concentration of APOC4 protein ≥ 5.094 ng / mL, it is judged as positive for small cell lung cancer.

[0047] Example 1

[0048] In this example, biomarker screening and diagnostic verification were carried out.

[0049] (1) Clinical sample recruitment

[0050] This study was approved by the Clinical Research Ethics Committee of the Affiliated Hospital of North China University of Science and Technology, and all participants signed written informed consent forms. From April 2024 to March 2025, samples of NSCLC and SCLC patients were prospectively collected. The inclusion criteria for patients were as follows: 1) Pathologically diagnosed as NSCLC or SCLC; 2) Not received chemotherapy or radiotherapy; 3) No history of other malignancies

[0051] (2) Sample collection

[0052] Peripheral blood samples of the subjects were collected using aseptic techniques, immediately sent to the laboratory after collection, left to stand at room temperature for 1 h, centrifuged at 3000 rpm for 10 min to separate the serum. The separated serum samples were transferred to an ultra-low temperature freezer at -80 °C for storage until use.

[0053] (3) Screening and external validation of diagnostic marker candidate proteins

[0054] Perform proteomic analysis on serum samples using UHPLC-MS / MS technology. The specific analysis conditions are as follows: 1) Liquid chromatography conditions: C18 chromatographic column. Mobile phase A is prepared from 100% water and 0.1% formic acid, and mobile phase B is prepared from 80% acetonitrile and 0.1% formic acid. The gradient elution program is 4% B from 0 - 0.2 min with a flow rate of 2.5 μL / min, which drops to 2.0 μL / min at 0.2 min; from 0.3 - 12.2 min, the flow rate is 1.5 μL / min, and the mobile phase gradient changes to 8% B at 0.3 min, 22.5% B at 7.5 min, and 35.0% B at 12.2 min; from 12.6 - 13.7 min, the flow rate is 2.5 μL / min, 55% B at 12.6 min, and 99% B at 13.0 min. 2) Mass spectrometry parameters: Electrospray ionization source (ESI), set the ion spray voltage to 1.9 kV, the ion transfer tube temperature to 290 °C, and adopt data-dependent acquisition mode. According to the results of serum proteomic analysis, screen out the proteins with significant differences between NSCLC and SCLC. For the differential proteins with a coverage of ≥50% in both groups of samples, further include independent samples from the Affiliated Hospital of North China University of Science and Technology (14 cases each of NSCLC and SCLC) for ELISA verification, and screen out the proteins with significant differences and consistent differential trends in both datasets.

[0055] (4) ROC curve evaluation of the diagnostic performance of candidate proteins

[0056] Using the expression levels of candidate proteins, draw ROC curves to evaluate the diagnostic performance of the screened key candidate proteins in differentiating NSCLC from SCLC, determine the optimal diagnostic threshold, and calculate its accuracy, sensitivity, specificity, and AUC value.

[0057] Experimental results

[0058] (1) Screening of diagnostic marker candidate proteins

[0059] Based on the serum proteomic results of 10 NSCLC patients and 10 SCLC patients, with Fold change > 2 and P < 0.05 as the differential protein screening conditions, a total of 17 differential proteins were screened. Subsequently, ELISA was used for quantitative verification of 8 differential proteins (SPP1, IgH, PZP, APOC2, APOC4, ACAN, ATP5F1A, and RRP1) with a coverage of ≥50% in both groups of samples. Among them, there is no detection kit for RRP1, so only the verification results of 7 differential proteins were obtained ( Figure 1 and Figure 2) The verification results showed that there were significant differences in the concentrations of four differential proteins, namely APOC2, APOC4, ACAN, and ATP5F1A, in the two groups of verification samples. Among them, APOC2 and APOC4 showed a consistent trend with the differential analysis results of the discovery set, both being upregulated in the SCLC group; while ACAN and ATP5F1A showed an opposite expression trend to the discovery set.

[0060] (2) Evaluation of the diagnostic performance of candidate proteins and determination of the optimal threshold

[0061] The diagnostic performance of two differential proteins, APOC2 and APOC4, for NSCLC and SCLC in the two datasets was evaluated using the ROC curve. The results showed that in the discovery set, the AUC value of the APOC2 protein was 0.72, and the AUC value of the APOC4 protein was 0.80. In the external validation cohort, as Figure 3 shown, the optimal threshold for the APOC2 protein was determined to be 9.226 ng / mL, that is, when APOC2 < 9.226 ng / mL, it was diagnosed as NSCLC, and when APOC2 ≥ 9.226 ng / mL, it was diagnosed as SCLC. Its diagnostic accuracy was 96%, sensitivity was 100%, specificity was 93%, and the AUC value was 0.99; the optimal threshold for the APOC4 protein was 5.094 ng / mL, that is, when APOC4 < 5.094 ng / mL, it was diagnosed as NSCLC, and when APOC4 ≥ 5.094 ng / mL, it was diagnosed as SCLC. Its diagnostic accuracy was 86%, sensitivity was 71%, specificity was 100%, and the AUC value was 0.88.

[0062] The AUC values of existing lung cancer classification biomarkers are generally < 0.85, which cannot meet the needs of clinical precision classification of lung cancer. In the present invention, the diagnostic efficiency of APOC2 and APOC4 candidate proteins for NSCLC and SCLC in the discovery set was relatively high (the AUCs were 0.72 and 0.80 respectively). In the external validation cohort, the optimal thresholds of APOC2 and APOC4 were determined to be 9.226 and 5.094 ng / mL respectively, showing good diagnostic performance (the AUCs were 0.99 and 0.88 respectively), indicating their reliability and practicality in the precision classification of lung cancer.

[0063] In summary, the biomarkers of the present invention can effectively distinguish non - small cell lung cancer and small cell lung cancer. The APOC2 protein and APOC4 protein had relatively high diagnostic efficiency for NSCLC and SCLC in the discovery set (the AUCs were 0.72 and 0.80 respectively). In the external validation cohort, the optimal thresholds of APOC2 and APOC4 were determined to be 9.226 and 5.094 ng / mL respectively, showing good diagnostic performance (the AUCs were 0.99 and 0.88 respectively), indicating their reliability and practicality in the precision classification of lung cancer.

[0064] The applicant declares that the present invention illustrates the detailed method of the present invention through 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 improvement to the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A serum protein biomarker for lung cancer typing, characterized in that, The serum protein biomarkers for lung cancer typing include: APOC2 protein and / or APOC4 protein.

2. The serum protein biomarker for lung cancer typing according to claim 1, characterized in that, The lung cancer includes non-small cell lung cancer and / or small cell lung cancer.

3. Use of the serum protein biomarker for lung cancer typing and / or its detection reagent according to claim 1 or 2 in the preparation of a product for lung cancer typing.

4. A kit for lung cancer typing, characterized in that, The kit includes a reagent for detecting the presence or expression level of the serum protein biomarker for lung cancer typing according to claim 1 or 2.

5. A device for lung cancer typing, characterized in that, The device includes a detection unit and an analysis unit; The detection unit is used to perform: detecting the expression level of APOC2 protein and / or APOC4 protein in a sample to be tested; The analysis unit is used to perform: judging whether the patient is a non-small cell lung cancer patient or a small cell lung cancer patient according to the expression level of APOC2 protein and / or APOC4 protein in the sample to be tested.

6. The device according to claim 5, characterized in that The sample to be tested includes serum.

7. The device according to claim 5 or 6, characterized in that, The criteria for judgment are: (1) When the concentration of APOC2 protein < 9.226 ng / mL, it is judged as positive for non-small cell lung cancer, and when the concentration of APOC2 protein ≥ 9.226 ng / mL, it is judged as positive for small cell lung cancer; or, (2) When the concentration of APOC4 protein < 5.094 ng / mL, it is judged as positive for non-small cell lung cancer, and when the concentration of APOC4 protein ≥ 5.094 ng / mL, it is judged as positive for small cell lung cancer.

8. A model for lung cancer typing, characterized in that, The input variable of the model for lung cancer typing is the concentration of APOC2 protein or the concentration of APOC4 protein, and the output variable is positive for non-small cell lung cancer or positive for small cell lung cancer.

9. The model according to claim 8, wherein The lung cancer typing includes non-small cell lung cancer and small cell lung cancer.

10. Use of the serum protein biomarker for lung cancer typing according to claim 1 or 2, the device for lung cancer typing according to any one of claims 5-7, or the model for lung cancer typing according to claim 8 or 9 in screening drugs for treating non-small cell lung cancer and / or small cell lung cancer.

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