Exhaled air condensate metabolite combination, kit and application in predicting lung adenocarcinoma metastasis

Abstract

The invention discloses an exhaled gas condensate metabolite combination, a kit and application of the exhaled gas condensate metabolite combination to prediction of lung adenocarcinoma metastasis, and the exhaled gas condensate metabolite combination comprises pentadecanoic acid, phosphocreatine, glycyl-L-glutamine, acetyl-L-homoserine, 2-amino-2-methyl-1, 3-propanediol, 2-amino-2-methyl-1, 3-propanediol, 2-amino-2-methyl-1, 3-propanediol, 2-amino-2-methyl-1, 3-propanediol, 2-amino-2-methyl-1, 3-propanediol, 2-methyl-1 The marker can be used as a biomarker combination for diagnosing whether metastatic lung adenocarcinoma exists or not, and has good sensitivity and specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of lung adenocarcinoma metastasis diagnosis, in particular to an exhaled breath condensate metabolite combination, a kit and application in predicting lung adenocarcinoma metastasis. BACKGROUND

[0002] Lung adenocarcinoma metastasis is a common extrapulmonary tuberculosis in clinic, with a high incidence, accounting for about 44.1% of the entire pleural disease. The incidence and mortality of lung cancer is one of the highest tumors in the world, and about 40% of lung cancer patients will develop malignant pleural effusion during the disease process. At present, due to the limitation of diagnostic techniques, the diagnosis rate of the two is very low. However, lung adenocarcinoma metastasis and malignant pleural effusion have completely different prognosis and treatment, and if not diagnosed and treated in time, it will directly affect the quality of life and survival of patients. Therefore, how to quickly and accurately differentiate between benign and malignant pleural effusion is the focus and difficulty of current clinical research.

[0003] At present, the main means for diagnosing and differentiating lung cancer metastasis in clinic include imaging examination and pathological examination. Among them, computed tomography (CT) is the most commonly used imaging method, which can clearly show the size, shape and location of the primary lung tumor, and is helpful for detecting metastases in other parts, such as liver, bone and other parts. However, the diagnostic accuracy of this method is limited by the level of the diagnostician and the precision of the instrument, and has limitations such as low sensitivity and strong subjectivity, which can easily lead to missed diagnosis or misdiagnosis, and cannot meet the needs of clinical work.

[0004] Therefore, it is of great clinical significance to develop and design a new, high-sensitivity lung adenocarcinoma metastasis diagnostic technique. SUMMARY

[0005] The present application provides an exhaled breath condensate metabolite combination and a kit for predicting lung adenocarcinoma metastasis, providing a new approach for clinical diagnosis.

[0006] In view of this, the scheme of the present application is: The first aspect of the present application is to propose the use of a biomarker combination in the preparation of a lung adenocarcinoma metastasis diagnostic product, wherein the biomarker combination comprises five metabolites, including pentadecanoic acid, phosphocreatine, glycyl-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol.

[0007] Further, the diagnostic product is selected from diagnostic reagents, chips, carriers or kits.

[0008] Further, the application comprises the following steps: 1) obtaining an exhaled breath or exhaled breath condensate sample of a subject individual; 2) determining absolute or relative quantitative values of each of the five metabolites in the sample from step 1).

[0009] A second aspect of the present application is to provide a kit comprising reagents for detecting the combination of metabolites in exhaled breath condensate, and / or a detection device, and / or a detection system; the combination of metabolites is pentadecanoic acid, creatine phosphate, glycyl-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol.

[0010] Further, the detection reagents include liquid chromatography and / or mass spectrometry detection reagents.

[0011] Further, the kit further comprises sample processing reagents for pretreating the exhaled breath condensate, including but not limited to removal of impurities.

[0012] Further, the kit further comprises a standard database of the combination of metabolites or software or chips thereof.

[0013] Further, the kit further comprises a reference line for reference and giving a prediction result.

[0014] A third aspect of the present application is to provide a lung adenocarcinoma metastasis detection system, comprising: a sample collection and processing unit for collecting exhaled breath or condensate thereof of a subject to be tested and processing to obtain a sample; a detection unit for detecting the level of each metabolite in the sample, the metabolite being pentadecanoic acid, creatine phosphate, glycyl-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol; a prediction unit for one of: i) obtaining a prediction or diagnosis result by comparing with a detection reference value according to the level of each metabolite in the detection unit; ii) obtaining a prediction or diagnosis result by combining and analyzing the level of each metabolite in the detection unit with database or known population sample data.

[0015] Further, for i), the level of each metabolite in the detection unit can be input into a regression formula and combined with the detection reference value to obtain the result, and the regression formula is: Prob = 1 / (1 + e - X ) where X = -4.43a + 2.72b + 0.49c - 3.34d + 1.10e + 23.63; In the formula, Prob is the probability of lung adenocarcinoma metastasis; a~e respectively correspond to the detected content of pentadecanoic acid, creatine phosphate, glycine-glutamine, acetylhomoserine, amino-methyl-propanediol; the prediction or diagnosis result is obtained by combining the detection value with the reference value in the embodiments of the present application. For example, in some embodiments, the reference value Cutoff=0.295, that is, when the Prob value is greater than 0.295, it is diagnosed as metastatic lung adenocarcinoma, and when it is less than or equal to 0.295, it is diagnosed as non-metastatic lung adenocarcinoma.

[0016] Compared with the prior art, the present application has the following beneficial effects: The present application provides that the exhaled breath condensate metabolites pentadecanoic acid, creatine phosphate, glycine-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol can be used in combination for the identification of lung adenocarcinoma metastasis patients. Through the marker combination involved in the present application, the diagnosis of lung adenocarcinoma metastasis has good sensitivity and specificity, and provides important guidance for non-invasive diagnosis and clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A bar chart showing the content change (represented by mean ± standard error) of pentadecanoic acid, creatine phosphate, glycine-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol in the exhaled breath condensate of non-metastatic and metastatic lung adenocarcinoma disease patients in different embodiments of the present application.

[0018] Figure 2 ROC curve for discriminating metastatic lung adenocarcinoma group in different embodiments of the present application. DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be described below in conjunction with preferred embodiments, obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0020] In the embodiments of the present application, the five small molecule metabolites in the exhaled breath condensate determined by screening are pentadecanoic acid, creatine phosphate, glycine-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol, which are important metabolites in the human body. The specific chemical information is as follows: Pentadecanoic acid (CAS No.: 1002-84-2), creatine phosphate (CAS No.: 67-07-2), glycyl-L-glutamine (CAS No.: 13115-71-4), acetyl-L-homoserine (CAS No.: 83592-11-4), 2-amino-2-methyl-1,3-propanediol (CAS No.: 115-69-5).

[0021] This invention combines the above five metabolites and applies them to the diagnosis of metastatic lung adenocarcinoma. The mass spectrometry targeted analysis ion information of this combination is shown in Table 1 below.

[0022] Table 1: Information on the Targeting Analysis of Five Combined Biomarkers

[0023] The method for detecting and predicting lung adenocarcinoma metastasis based on the above five metabolite combinations as biomarkers is as follows: (1) Pretreatment of exhaled breath condensate samples The exhaled breath condensate sample was thawed at 4℃. 100 µL of the condensate was taken and 100 µL of 10-80% (v / v) acetonitrile methanol extraction solution was added to precipitate the protein. The sample was vortexed for 30-90 s, allowed to stand for 15-30 mins, and then centrifuged at 10000-14000×g for 10-15 mins at 4℃. The supernatant was collected and freeze-dried. 100 µL of 10-80% (v / v) methanol aqueous solution was added and the sample was centrifuged at 10000-14000×g for 10-15 mins at 4℃. The supernatant was collected for LC-MS / MS analysis.

[0024] (2) Targeted detection of target metabolites using liquid chromatography-mass spectrometry

[0025] The separation system was ultra-high performance liquid chromatography (UHPLC), using a C8 column and a mobile phase flow rate of 0.2–0.35 m / s.

[0026] The flow rate was set at 0.2-1.0 ml / min, with a column temperature of 40-60°C and an injection volume of 5-10 μL (a C18 column can also be used, with a column temperature of 40-60°C and a flow rate of 0.2-1.0 ml / min). The eluent consisted of phase A (0.1% v / v formic acid in aqueous solution) and phase B (0.1% v / v formic acid in acetonitrile solution) (or 0-0.1% v / v formic acid in aqueous solution and acetonitrile (methanol) solution). The detector used was a high-sensitivity single quadrupole mass spectrometer or a tandem quadrupole mass spectrometer (Q-trap MS, QQQ MS), with positive ion detection. The targeted ion information for this combination is shown in Table 1.

[0027] (3) Judgment model based on mass spectrometry detection data: The obtained data were analyzed using binary logistic regression, and the regression equation obtained from the constructed model is shown in Equation 1: Prob = 1 / (1+e) - X (1) Where X = -4.43a + 2.72b + 0.49c - 3.34d + 1.10e + 23.63 The exhaled breath condensate sample was taken from the subject and the extract was tested to detect pentadecanoic acid a, phosphocreatine b, glycine-glutamine c, acetylhomoserine d, and amino-methyl-propylene glycol e.

[0028] Exhaled breath condensate samples were collected from the subjects, and metabolites were extracted. The contents of pentadecanoic acid, phosphocreatine, glycyl-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol in the exhaled breath condensate samples were quantified by mass spectrometry. Prob represents the probability of lung adenocarcinoma metastasis, and Cutoff = 0.295. That is, when the Prob value is greater than 0.295, the diagnosis is metastatic lung adenocarcinoma.

[0029] The established model has good discriminative ability against metastatic lung adenocarcinoma (see...). Figure 2 In Example 1, the combined biomarker had an AUC of 0.884, a sensitivity of 80.0%, and a specificity of 83.0%; in Example 2, the combined biomarker had an AUC of 0.895, a sensitivity of 93.3%, and a specificity of 66.7%. This indicates that the five combined biomarkers have the potential to diagnose metastatic lung adenocarcinoma.

[0030] Example 1

[0031] 1. Before collecting exhaled breath condensate samples, volunteers must sign an informed consent form.

[0032] Inclusion criteria for patients with metastatic lung adenocarcinoma: imaging examination reveals lung nodules or space-occupying lesions and biopsy pathology reveals tumor cells; there is exudative pleural effusion, and tumor cells are found in the exfoliated cells of the pleural effusion; and / or pleural histopathology confirms pleural metastasis of tumor.

[0033] Inclusion criteria for non-metastatic lung adenocarcinoma patients: imaging examination reveals lung nodules or space-occupying lesions and biopsy pathology reveals tumor cells; there is exudative pleural effusion, and tumor cells are found in the exfoliated cells of the pleural effusion.

[0034] Exhaled breath condensate samples were collected under the same conditions from 47 patients with non-metastatic lung adenocarcinoma and 35 patients with metastatic lung adenocarcinoma. 50 ml of exhaled breath condensate was collected from each patient before any treatment was administered upon admission. The exhaled breath condensate was separated within half an hour and stored in a -80°C refrigerator for future testing.

[0035] 2. Analytical Methods

[0036] 2.1 Pretreatment of Exhaled Breath Claritrate Samples

[0037] The exhaled breath condensate sample was thawed at 4℃, and 100 µL was taken. 400 µL of methanol extract was added to precipitate the protein. The sample was vortexed for 30-90 s, allowed to stand for 15-30 mins, and then centrifuged at 10000-14000×g for 10-15 mins at 4℃. The supernatant was collected and freeze-dried. 50 µL of 10-20% (v / v) methanol aqueous solution was added, and the sample was centrifuged at 12000×g for 15 mins at 4℃. The supernatant was collected for LC-MS / MS analysis, and the contents of six metabolites were calculated by extracting ion chromatograms.

[0038] 2.2 Ultra-high performance liquid chromatography-mass spectrometry analysis

[0039] (1) Liquid chromatography conditions: The chromatograph was a Nexera LC-30AD ultra-high performance liquid chromatograph (Shimadzu, Kyoto, Japan); the chromatographic column was a Waters ACQUITY UPLC @ BEH C8 (1.7um, 2.1mm X100mm) (Waters, Ireland); the mobile phase A was a 0.1% (v / v) formic acid aqueous solution and the mobile phase B was a 0.1% (v / v) formic acid acetonitrile solution; the elution gradient was 2% B phase from 0 to 1 min, linearly changing to 80% B phase from 1 min to 20 min, linearly changing to 100% B phase from 20 min to 25 min, holding the phase for 4 min, and linearly decreasing to 2% B phase within 0.1 min and holding for 1 mins; the column temperature was 50℃; the mobile phase flow rate was 0.35 mL / min; and the injection volume was 10 µL.

[0040] (2) Mass spectrometry conditions: The mass spectrometer was a quadrupole-ion trap mass spectrometer (Q-Trap 5500 MS) (AB SCIEX, Framingham, MA, USA); positive ion mode was used for detection; the curtain gas, GAS1, and GAS2 were set to 50 arbitrary units, 35 arbitrary units, and 35 arbitrary units, respectively; the ion source temperature was 500 ℃, and the spray voltage was 5.0 kV.

[0041] 2.3 Exhaled Breath Clarification Test Results and Auxiliary Diagnostic Methods

[0042] For the malignant pleurisy group, pentadecanoic acid, creatine phosphate, glycyl-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol showed significant changes (see [link to relevant data]). Figure 1A). Simultaneously, the levels of each biomarker were substituted into the regression equation of Equation 1 to calculate the probability. The cutoff value used was 0.295, meaning that a probability greater than 0.295 for the combined biomarker was considered metastatic lung adenocarcinoma. Using this method, 36 patients were ultimately diagnosed with metastatic lung adenocarcinoma. Of the 35 patients diagnosed with metastatic lung cancer by pathological biopsy, 28 were diagnosed with metastatic lung cancer. Of the 47 patients diagnosed with non-metastatic lung cancer by pathological biopsy, 35 were diagnosed with non-metastatic lung cancer. The AUC of the combined biomarker was 0.884 (see...). Figure 2 A), with relatively high sensitivity and specificity, at 80.0% and 83.0% respectively.

[0043] Example 2

[0044] To verify the stability and reliability of the combined biomarkers, external independent samples were used for validation. The analytical methods, instruments, and reagents used in this experiment were completely consistent with those in Example 1.

[0045] 1. Before collecting exhaled breath condensate samples, volunteers must sign an informed consent form.

[0046] Inclusion criteria for patients with metastatic pulmonary adenocarcinoma: imaging examination reveals pulmonary nodules or space-occupying lesions and biopsy pathology reveals tumor cells; there is exudative pleural effusion, and tumor cells are found in the exfoliated cells of the pleural effusion; and / or pleural histopathology confirms pleural metastasis of tumor.

[0047] Exhaled breath condensate samples were collected under the same conditions from 33 patients with non-metastatic lung adenocarcinoma and 15 patients with metastatic lung adenocarcinoma. 50 ml of exhaled breath condensate was collected from each patient before any treatment was administered upon admission. The exhaled breath condensate was separated within half an hour and stored in a -80°C refrigerator for future testing.

[0048] 2. Exhaled breath condensate test results and auxiliary diagnostic methods

[0049] For the malignant pleurisy group, pentadecanoic acid, creatine phosphate, glycyl-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol showed significant changes (see [link to relevant data]). Figure 1 B). Simultaneously, the levels of each biomarker were input into the regression equation to calculate the probability. A cutoff value of 0.295 was used, meaning that a probability greater than 0.295 for the combined biomarker was considered metastatic lung adenocarcinoma. Using this method, 25 patients were ultimately diagnosed with metastatic lung adenocarcinoma. Of the 15 patients diagnosed with metastatic lung cancer via pathological biopsy, 14 were diagnosed with metastatic lung cancer. Of the 23 patients diagnosed with non-lung cancer risk via pathological biopsy, 22 were diagnosed with non-lung cancer metastasis. The AUC of the combined biomarker was 0.895 (see...). Figure 2(B) The sensitivity and specificity are also relatively high, at 93.3% and 66.7%, respectively.

[0050] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of biomarker combinations in the preparation of diagnostic products for lung adenocarcinoma metastasis, characterized in that, The biomarker combination consists of five metabolites, including pentadecanoic acid, creatine phosphate, glycyl-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol.

2. The application as described in claim 1, characterized in that, The diagnostic products are selected from diagnostic reagents, chips, carriers, or kits.

3. The application as described in claim 1, characterized in that, The application includes the following steps: 1) Obtain exhaled breath or exhaled breath condensate samples from individual subjects; 2) Determine the absolute or relative quantitative values ​​of each of the five metabolites in the sample from step 1).

4. A reagent kit, characterized in that, It contains a reagent for detecting a combination of metabolites in exhaled condensate, and / or a detection device and / or a detection system; the combination of metabolites is: pentadecanoic acid, creatine phosphate, glycyl-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol.

5. The kit according to claim 4, characterized in that, The detection reagents include liquid chromatography and / or mass spectrometry detection reagents.

6. The kit according to claim 4, characterized in that, It also includes sample processing reagents.

7. The kit according to claim 4, characterized in that, It also includes a standard database of the metabolite combination or its software or chip.

8. The kit according to claim 4, characterized in that, It also includes reference lines.

9. A lung adenocarcinoma metastasis detection system, characterized in that, include: Sample collection and processing unit: used to collect the exhaled breath or condensate of the subject and process it to obtain a sample; Detection unit: used to detect the level of each metabolite in the sample, wherein the metabolites are pentadecanoic acid, phosphocreatine, glycyl-L-glutamine, acetyl-L-homoserine, and 2-amino-2-methyl-1,3-propanediol; Prediction unit, used for one of the following: i) Based on the level of each metabolite detected by the detection unit, a predictive or diagnostic result is obtained by comparing it with a detection reference value; ii) Based on the level of each metabolite detected by the detection unit, the results are calculated and analyzed by combining the data with database or known population sample data to obtain a prediction or diagnosis.

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