Biomarker for distinguishing lung cancer and pneumonia and application thereof

Abstract

The invention discloses a biomarker for distinguishing lung cancer and pneumonia and application of the biomarker. The biomarker for distinguishing lung cancer and pneumonia comprises trans-oleic acid. The biomarker can effectively distinguish lung cancer and pneumonia, and for NSCLC diagnosis, the area AUC below an operation characteristic curve of a subject is 0.79, the sensitivity is 70%, and the specificity is 90%; for SCLC diagnosis, the AUC value is 0.75, the sensitivity is 60%, and the specificity is 90%, meanwhile, in an external verification set, the metabolic difference trend of the trans-oleic acid in lung cancer and pneumonia patients is consistent with the result of a discovery set, the AUC value of the trans-oleic acid when NSCLC and PNA are diagnosed is 0.74, and the AUC value of the trans-oleic acid when SCLC and PNA are diagnosed is 0.92.

Description

Technical Field

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

[0002] Lung cancer, as one of the malignant tumors with the highest incidence and mortality globally, has posed a major public health challenge. Lung cancer is mainly divided into two pathological subtypes: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Although low-dose computed tomography (LDCT) screening has significantly improved the early detection rate of lung cancer and can reduce the lung cancer mortality rate by about 20%, it still has relatively high false positive and false negative problems. Tumor biomarker detection, with its advantages of specificity, minimally invasive nature, convenience, economy, etc., plays a key role in the early detection, diagnosis, treatment monitoring, and prognosis evaluation of malignant tumors.

[0003] Metabolic reprogramming is one of the important characteristics of cancer and is closely related to the occurrence, development, and treatment of tumors. A large number of studies have shown that cancer cells reshape their metabolic networks to meet the bioenergetic and biosynthetic demands of rapid proliferation. These metabolic alterations not only promote the malignant progression of tumors but also provide new biomarkers and potential targets for cancer diagnosis and treatment. Bronchoalveolar lavage fluid (BALF), as a biological sample directly collected from the pulmonary lesion area, can more precisely reflect the metabolomic characteristics of the local lung microenvironment, thereby providing superior diagnostic specificity and sensitivity. Screening biomarkers based on BALF metabolites is expected to improve the early diagnosis rate of lung cancer and overall enhance the prevention and treatment level of tumors.

[0004] Existing diagnostic markers have many limitations. Taking carcinoembryonic antigen (CEA), which is widely used clinically, as an example, it can be elevated in various malignant tumors and inflammatory diseases, resulting in insufficient diagnostic specificity. Developing specific biomarkers that can effectively distinguish malignant tumors from inflammatory diseases is of great clinical significance for improving the accuracy of early diagnosis of lung cancer. Summary of the Invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a biomarker for distinguishing lung cancer and pneumonia and its application, which is screened based on liquid chromatography-mass spectrometry (LC-MS). Specifically, the present invention performs untargeted metabolomics analysis on bronchoalveolar lavage fluid samples of patients with NSCLC, SCLC, and pneumonia (PNA), screens out metabolites with significant differences between the NSCLC and PNA groups and the SCLC and PNA groups, and further verifies them through targeted metabolomics, and finds that elaidic acid can be used as a specific biomarker for the diagnosis of lung cancer. This metabolite shows a significant downward trend in the bronchoalveolar lavage fluid of NSCLC and SCLC patients and has excellent diagnostic performance.

[0006] To achieve the object of the present invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a biomarker for distinguishing lung cancer and pneumonia, and the biomarker for distinguishing lung cancer and pneumonia includes: elaidic acid.

[0008] The biomarker of the present invention can effectively distinguish lung cancer and pneumonia. For the diagnosis of NSCLC, the area under the receiver operating characteristic curve AUC is 0.79, the sensitivity is 70%, and the specificity is 90%; for the diagnosis of SCLC, the AUC value is 0.75, the sensitivity is 60%, and the specificity is 90%. At the same time, in the external validation set, the metabolic difference trend of elaidic acid in lung cancer and pneumonia patients is consistent with the results of the discovery set, and the AUC value for diagnosing NSCLC and PNA is 0.74, and the AUC value for diagnosing SCLC and PNA is 0.92.

[0009] Preferably, the lung cancer includes non-small cell lung cancer and / or small cell lung cancer.

[0010] In a second aspect, the present invention provides the use of the biomarker for distinguishing lung cancer and pneumonia described in the first aspect and / or its detection reagent in the preparation of a product for distinguishing lung cancer and pneumonia.

[0011] In a third aspect, the present invention provides a kit for distinguishing lung cancer and pneumonia, and the kit includes a reagent for detecting the presence or expression level of the biomarker for distinguishing lung cancer and pneumonia described in the first aspect.

[0012] In a fourth aspect, the present invention provides a device for distinguishing lung cancer and pneumonia, and the device includes a detection unit and an analysis unit;

[0013] The detection unit is used to perform the following: detecting the concentration value of trans-oleic acid in the sample to be tested;

[0014] The analysis unit is used to perform the following: judging whether the patient is a lung cancer patient or a pneumonia patient according to the concentration value of trans-oleic acid in the sample to be tested.

[0015] Preferably, the sample to be tested includes bronchoalveolar lavage fluid, preferably the bronchoalveolar lavage fluid of a lung cancer or pneumonia patient.

[0016] Preferably, the criteria for judgment are as follows:

[0017] (1) When differentiating non-small cell lung cancer and pneumonia, if the concentration of trans-oleic acid in the sample to be tested is <0.61 ng / mL, it is judged as positive for non-small cell lung cancer; if the concentration of trans-oleic acid in the sample to be tested is ≥0.61 ng / mL, it is judged as positive for pneumonia;

[0018] (2) When differentiating small cell lung cancer and pneumonia, if the concentration of trans-oleic acid in the sample to be tested is <0.566 ng / mL, it is judged as positive for small cell lung cancer; if the concentration of trans-oleic acid in the sample to be tested is ≥0.566 ng / mL, it is judged as positive for pneumonia.

[0019] In a fifth aspect, the present invention provides a model for differentiating lung cancer and pneumonia. The input variable of the model for differentiating lung cancer and pneumonia is the concentration value of trans-oleic acid in bronchoalveolar lavage fluid, and the output variable is positive for lung cancer or positive for pneumonia.

[0020] Preferably, the lung cancer includes non-small cell lung cancer and / or small cell lung cancer.

[0021] Preferably, the criteria for judgment of the output variable are as follows:

[0022] (1) When differentiating non-small cell lung cancer and pneumonia, if the concentration of trans-oleic acid in the sample to be tested is <0.61 ng / mL, it is judged as positive for non-small cell lung cancer; if the concentration of trans-oleic acid in the sample to be tested is ≥0.61 ng / mL, it is judged as positive for pneumonia;

[0023] (2) When differentiating small cell lung cancer and pneumonia, if the concentration of trans-oleic acid in the sample to be tested is <0.566 ng / mL, it is judged as positive for small cell lung cancer; if the concentration of trans-oleic acid in the sample to be tested is ≥0.566 ng / mL, it is judged as positive for pneumonia.

[0024] In a sixth aspect, the present invention provides the use of the biomarker for differentiating lung cancer and pneumonia according to the first aspect, the device for differentiating lung cancer and pneumonia according to the fourth aspect, or the model for differentiating lung cancer and pneumonia according to the fifth aspect in screening drugs for treating lung cancer and / or pneumonia.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention for the first time discovers the biomarker trans-oleic acid for diagnosing lung cancer, which can achieve accurate diagnosis of lung cancer and pneumonia. Compared with pneumonia patients, the level of trans-oleic acid in the bronchoalveolar lavage fluid of NSCLC and SCLC patients is significantly reduced, having significant diagnostic value;

[0027] (2) In the discovery set of the present invention, for the diagnosis of NSCLC, the area under the Receiver Operating Characteristic Curve (ROC) is 0.79, the sensitivity is 70%, and the specificity is 90%; for the diagnosis of SCLC, the AUC value is 0.75, the sensitivity is 60%, and the specificity is 90%. At the same time, in the external validation set, the metabolic difference trend of trans-oleic acid in lung cancer and pneumonia patients is consistent with the results of the discovery set, and the AUC value for diagnosing NSCLC and PNA is 0.74, and the AUC value for diagnosing SCLC and PNA is 0.92. Trans-oleic acid has high sensitivity and specificity in distinguishing lung cancer and pneumonia patients, and shows good consistency in external validation. It has good reliability and clinical application potential as a lung cancer diagnostic biomarker. Description of the Drawings

[0028] Figure 1 It is a volcano plot of metabolite difference analysis between NSCLC and PNA, and SCLC and PNA;

[0029] Figure 2 It is a bubble plot of KEGG pathway enrichment of differential metabolites between NSLC and PNA, and SCLC and PNA;

[0030] Figure 3A It is an ROC curve graph of differential metabolites for distinguishing NSCLC and PNA;

[0031] Figure 3B It is an ROC curve graph of differential metabolites for distinguishing SCLC and PNA;

[0032] Figure 4 It is a graph of the expression level of trans-oleic acid in three groups of disease samples in the validation cohort;

[0033] Figure 5 It is an ROC curve graph of trans-oleic acid for distinguishing NSCLC and PNA, and SCLC and PNA in the validation cohort. Detailed Embodiments

[0034] To further illustrate the technical means adopted by the present invention and its effects, 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.

[0035] For those not specifying specific techniques or conditions in the embodiments, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0036] In a specific embodiment of the present invention, a kit for distinguishing lung cancer and pneumonia is provided. The kit includes reagents for detecting the presence or expression level of the biomarker for distinguishing lung cancer and pneumonia described in the present invention, such as any one or a combination of at least two of an ELISA detection kit (including a monoclonal antibody pair specifically recognizing trans-oleic acid), a chemiluminescence detection reagent (including a trans-oleic acid oxidase-linked reaction system), or an immunochromatographic test strip (coated with an anti-trans-oleic acid antibody).

[0037] In a specific embodiment of the present invention, a device for distinguishing lung cancer and pneumonia is provided. The device includes a detection unit and an analysis unit;

[0038] The detection unit is used to perform the following: detecting the concentration value of trans-oleic acid in a sample to be tested;

[0039] The analysis unit is used to perform the following: judging whether the patient is a lung cancer patient or a pneumonia patient according to the concentration value of trans-oleic acid in the sample to be tested.

[0040] The sample to be tested includes bronchoalveolar lavage fluid, preferably bronchoalveolar lavage fluid of a lung cancer or pneumonia patient.

[0041] The criteria for judgment are as follows:

[0042] (1) When distinguishing non-small cell lung cancer and pneumonia, if the concentration of trans-oleic acid in the sample to be tested < 0.61 ng / mL, it is judged as positive for non-small cell lung cancer; if the concentration of trans-oleic acid in the sample to be tested ≥ 0.61 ng / mL, it is judged as positive for pneumonia;

[0043] (2) When distinguishing small cell lung cancer and pneumonia, if the concentration of trans-oleic acid in the sample to be tested < 0.566 ng / mL, it is judged as positive for small cell lung cancer; if the concentration of trans-oleic acid in the sample to be tested ≥ 0.566 ng / mL, it is judged as positive for pneumonia.

[0044] In a specific embodiment of the present invention, a model for distinguishing lung cancer from pneumonia is provided. The input variable of the model for distinguishing lung cancer from pneumonia is the concentration value of trans-oleic acid in bronchoalveolar lavage fluid, and the output variable is positive for lung cancer or positive for pneumonia.

[0045] The lung cancer includes non-small cell lung cancer and / or small cell lung cancer.

[0046] The criteria for judgment are as follows:

[0047] (1) When distinguishing non-small cell lung cancer from pneumonia, if the concentration of trans-oleic acid in the test sample is <0.61 ng / mL, it is judged as positive for non-small cell lung cancer; if the concentration of trans-oleic acid in the test sample is ≥0.61 ng / mL, it is judged as positive for pneumonia.

[0048] (2) When distinguishing small cell lung cancer from pneumonia, if the concentration of trans-oleic acid in the test sample is <0.566 ng / mL, it is judged as positive for small cell lung cancer; if the concentration of trans-oleic acid in the test sample is ≥0.566 ng / mL, it is judged as positive for pneumonia.

[0049] Example 1

[0050] In this example, biomarker screening and diagnostic evaluation are carried out.

[0051] (1) Clinical sample recruitment

[0052] 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, SCLC and PNA patients were prospectively collected. The inclusion criteria for patients were as follows: NSCLC / SCLC patients were pathologically diagnosed; PNA patients were diagnosed by imaging and etiology, excluding those with concomitant lung tumors.

[0053] (2) Sample collection

[0054] Bronchoalveolar lavage fluid samples of 10 NSCLC patients, 10 SCLC patients and 10 PNA patients were collected and stored at -80 °C in ultra-low temperature until use.

[0055] (3) Screening of candidate metabolites for diagnostic markers

[0056] Non-targeted metabolomics analysis of bronchoalveolar lavage fluid was performed using LC-MS technology, and the specific analysis conditions were as follows: 1) Liquid chromatography conditions: C18 chromatographic column, mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was methanol; the gradient elution program was 2% B from 0 to 1.5 min, increased to 85% B from 1.5 to 3 min, increased to 100% B from 3 to 10 min, decreased to 2% B from 10 to 12 min, column temperature was 40 °C, and the flow rate was 0.2 mL / min; 2) Mass spectrometry parameters: electrospray ionization source (ESI), positive / negative ion mode, and the scanning range was selected as m / z 100-1500. Metabolites with significant differences in NSCLC and SCLC patients compared with PNA patients were screened according to the metabolomics results of bronchoalveolar lavage fluid, and key metabolites related to the pathway were screened by combining the key pathways enriched by differential metabolites.

[0057] (4) ROC curve to evaluate the diagnostic performance of candidate metabolites

[0058] Using the relative expression levels of candidate metabolites, ROC curves were plotted to evaluate the diagnostic performance of the selected key candidate metabolites in differentiating NSCLC from PNA and SCLC from PNA, and their AUC, sensitivity, and specificity were calculated.

[0059] (5) External cohort to validate the diagnostic value of metabolites from bronchoalveolar lavage fluid

[0060] Independent samples from the Affiliated Hospital of North China University of Science and Technology (10 cases each of NSCLC, SCLC, and PNA) were further included to validate the diagnostic candidate metabolites. Basic information and clinicopathological data of the patients, including gender, age, tumor stage, metastasis status, etc., were collected. Targeted metabolomics detection of the diagnostic candidate metabolites from bronchoalveolar lavage fluid was performed using LC-MS, the expression differences of the diagnostic candidate metabolites in NSCLC and PNA, SCLC and PNA patients were calculated, and ROC curves were plotted based on the metabolite expression levels to calculate AUC to evaluate their diagnostic performance.

[0061] Experimental results

[0062] (1) Screening of candidate metabolites for diagnostic markers

[0063] According to the metabolomics results of bronchoalveolar lavage fluid from 10 NSCLC patients, 10 SCLC patients, and 10 PNA patients, with VIP > 1.0, P < 0.05 and FC > 1.2 or FC < 0.833 as the screening criteria, a total of 217 differential metabolites were screened between the NSCLC and PNA groups, of which 87 were significantly up-regulated in the NSCLC group and 130 were significantly up-regulated in the PNA group; a total of 227 differential metabolites were screened between the SCLC and PNA groups, of which 61 were significantly up-regulated in SCLC and 166 were significantly up-regulated in PNA (Figure 1 ) KEGG pathway enrichment analysis of these differential metabolites found that the differential metabolites between the two disease comparison groups were significantly enriched in the biosynthesis pathway of unsaturated fatty acids ( Figure 2 ). Therefore, 97 metabolites with significant differences between NSCLC and PNA, and between SCLC and PNA were screened out, and 5 differential unsaturated fatty acid metabolites (oleoylethanolamide, adrenic acid, docosahexaenoic acid, trans-oleic acid, (+ / -)8(9)-DiHETE) were selected as diagnostic candidate metabolites.

[0064] (2) Evaluation of the diagnostic performance of diagnostic candidate metabolites

[0065] ROC curves were plotted for the 5 screened diagnostic candidate metabolites. As can be seen from Figure 3A and Figure 3B , the five diagnostic candidate metabolites had good diagnostic performance for NSCLC vs PNA and SCLC vs PNA, and their AUC values were all ≥0.75.

[0066] (3) Differential analysis of diagnostic candidate metabolites in the external validation set

[0067] To verify the clinical applicability of the candidate metabolites as lung cancer diagnostic biomarkers, 30 eligible subjects (10 NSCLC, 10 SCLC, 10 PNA) were collected again from the Affiliated Hospital of North China University of Science and Technology. Differential analysis of the candidate metabolites between groups showed that the level of trans-oleic acid was significantly decreased in the NSCLC and SCLC groups compared with the PNA group ( Figure 4 ).

[0068] (4) Evaluation of the diagnostic performance of candidate metabolites for lung cancer

[0069] Given that the above results support that trans-oleic acid in bronchoalveolar lavage fluid may be used as a biomarker for lung cancer diagnosis, it is necessary to further evaluate its feasibility in clinical practice. The diagnostic performance of trans-oleic acid in the validation cohort was evaluated using the ROC curve, and the optimal diagnostic threshold was determined. The results are as Figure 5As shown, when diagnosing NSCLC and PNA with trans-oleic acid, the threshold is 0.61 ng / mL. That is, when the trans-oleic acid < 0.61 ng / mL, it is diagnosed as NSCLC, and when the trans-oleic acid ≥ 0.61 ng / mL, it is diagnosed as PNA. Its diagnostic accuracy is 70%, sensitivity is 60%, specificity is 80%, and the AUC value is 0.74. When diagnosing SCLC and PNA with trans-oleic acid, the threshold is 0.566 ng / mL. That is, when the trans-oleic acid < 0.566 ng / mL, it is diagnosed as SCLC, and when the trans-oleic acid ≥ 0.566 ng / mL, it is diagnosed as PNA. Its diagnostic accuracy is 90%, sensitivity is 80%, specificity is 100%, and the AUC value is 0.92, indicating the diagnostic value of trans-oleic acid in bronchoalveolar lavage fluid for lung cancer.

[0070] Existing diagnostic markers have the problem of insufficient diagnostic specificity. Therefore, it is crucial to accurately identify lung cancer and pneumonia samples. For the diagnosis of NSCLC and PNA, SCLC and PNA in the discovery cohort, trans-oleic acid in bronchoalveolar lavage fluid has relatively high diagnostic efficiency (AUCs are 0.79 and 0.75 respectively). In the validation cohort, the optimal thresholds of trans-oleic acid for the diagnosis of NSCLC and PNA, SCLC and PNA were determined to be 0.61 and 0.566 ng / mL respectively, and both showed good diagnostic performance (AUCs are 0.74 and 0.92 respectively), indicating the reliability of trans-oleic acid in the diagnosis of lung cancer and pneumonia.

[0071] In summary, the biomarker of the present invention can effectively distinguish lung cancer and pneumonia. For the diagnosis of NSCLC, the area under the receiver operating characteristic curve (AUC) is 0.79, sensitivity is 70%, and specificity is 90%. For the diagnosis of SCLC, the AUC value is 0.75, sensitivity is 60%, and specificity is 90%. At the same time, in the external validation set, the metabolic difference trend of trans-oleic acid in lung cancer and pneumonia patients is consistent with the results of the discovery set, and the AUC value for diagnosing NSCLC and PNA is 0.74, and the AUC value for diagnosing SCLC and PNA is 0.92.

[0072] The applicant declares that the present invention uses the above embodiments to illustrate the detailed method of the present invention, 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 of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A biomarker for differentiating lung cancer from pneumonia, characterized in that, The biomarker for differentiating lung cancer from pneumonia includes: trans-oleic acid.

2. The biomarker for differentiating lung cancer from pneumonia 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 biomarker for differentiating lung cancer from pneumonia as claimed in claim 1 or 2 and / or its detection reagent in the preparation of a product for differentiating lung cancer from pneumonia.

4. A kit for differentiating lung cancer from pneumonia, characterized in that, The kit includes a reagent for detecting the presence or expression level of the biomarker for differentiating lung cancer from pneumonia as claimed in claim 1 or 2.

5. A device for differentiating lung cancer from pneumonia, characterized in that, The device includes a detection unit and an analysis unit; The detection unit is used to perform including: detecting the concentration value of trans-oleic acid in a sample to be tested; The analysis unit is used to perform including: judging whether the patient is a lung cancer patient or a pneumonia patient according to the concentration value of trans-oleic acid in the sample to be tested.

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

7. The device according to claim 5 or 6, characterized in that, The criteria for the judgment are as follows: (1) When differentiating non-small cell lung cancer from pneumonia, if the concentration of trans-oleic acid in the sample to be tested < 0.61 ng / mL, it is judged as positive for non-small cell lung cancer; if the concentration of trans-oleic acid in the sample to be tested ≥ 0.61 ng / mL, it is judged as positive for pneumonia; (2) When differentiating small cell lung cancer from pneumonia, if the concentration of trans-oleic acid in the sample to be tested < 0.566 ng / mL, it is judged as positive for small cell lung cancer; if the concentration of trans-oleic acid in the sample to be tested ≥ 0.566 ng / mL, it is judged as positive for pneumonia.

8. A model for differentiating lung cancer from pneumonia, characterized in that, The input variable of the model for differentiating lung cancer from pneumonia is the concentration value of trans-oleic acid in bronchoalveolar lavage fluid, and the output variable is positive for lung cancer or positive for pneumonia.

9. The model according to claim 8, characterized in that, The lung cancer includes non-small cell lung cancer and / or small cell lung cancer.

10. Use of the biomarker for differentiating lung cancer from pneumonia as claimed in claim 1 or 2, the device for differentiating lung cancer from pneumonia as claimed in any one of claims 5 - 7, or the model for differentiating lung cancer from pneumonia as claimed in claim 8 or 9 in the screening of drugs for treating lung cancer and / or pneumonia.

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