Exhaled air condensate metabolite combination, kit and method for predicting invasive lung adenocarcinoma

Abstract

The invention relates to an exhaled air condensate metabolite composition, a kit and a method for predicting invasive lung adenocarcinoma. The expired gas condensate metabolite composition for predicting the invasive lung adenocarcinoma comprises lysyl-valine, lysophosphatidyl glycerol (19: 0), vanillyl-glucose, hydroxyproline-threonine, phenylalanine, dihydroxy benzophenone, palmitoyl glycine, triglyceride (43: 0), monobutyl phthalate, hydroxyphenyl propionic acid, oleoyl glycine and dodecanedioic acid. The preparation method comprises the following steps: adding 4-chlorophenylalanine and The kit for predicting the invasive lung adenocarcinoma comprises an internal standard substance required for detecting the metabolite combination. The combined marker provided by the invention has the characteristics of high sensitivity and high specificity on the diagnosis of the invasive lung adenocarcinoma.

Description

Technical Field

[0001] The present invention relates to a method for predicting invasive lung adenocarcinoma, and in particular to an exhaled breath condensate metabolite combination, a kit and a method for predicting invasive lung adenocarcinoma. Background Art

[0002] Invasive lung adenocarcinoma is a common extrapulmonary tuberculosis in clinical practice, with a high incidence, accounting for about 44.1% of the total pleural diseases, while the incidence of malignant pleurisy in my country is as high as 900,000 per year, accounting for about 9% of the total incidence in the world. The incidence and mortality of lung cancer are among the highest in the world, and about 40% of lung cancer patients will develop malignant pleural effusion during the course of the disease. At present, due to the limitations of diagnostic technology, the diagnosis rate of both is very low. However, the prognosis and treatment of invasive lung adenocarcinoma and malignant pleurisy are completely different. If they are not diagnosed and treated in time, it will directly affect the patient's quality of life and survival. Therefore, how to quickly and accurately distinguish between benign and malignant pleurisy is a hot spot and difficulty in current clinical research.

[0003] At present, the methods for clinical diagnosis and identification of invasive lung adenocarcinoma and malignant pleurisy include clinical manifestations, laboratory results of thoracentesis, pleural biopsy, bronchoscopy, surgical biopsy, etc. The most commonly used method is cytological examination by thoracentesis. If tumor cells are found in pleural effusion, it has diagnostic value for malignant pleurisy. However, due to the complex cell composition in pleural effusion, it is sometimes difficult to distinguish tumor cells from inflammatory mesothelial cells, macrophages, etc., and it is also affected by factors such as the level of the diagnostician and sample collection. Its sensitivity is low (about 40%-60%) and its subjectivity is strong, which can easily lead to missed diagnosis or misdiagnosis. The diagnosis of invasive lung adenocarcinoma mainly includes biochemical examination of exhaled breath condensate, tuberculin skin test (TST), bacteriological examination of pleural effusion (acid-fast staining smear and Mycobacterium tuberculosis culture), pleural tissue pathological biopsy, etc. However, the above-mentioned examination methods have low specificity and sensitivity, and some methods are highly invasive, high-risk, or time-consuming, and cannot meet the needs of clinical work. Therefore, the development of new and highly sensitive pleurisy diagnosis technology is of great clinical significance. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide an exhaled breath condensate metabolite combination, a kit and a method for predicting invasive lung adenocarcinoma.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] A panel of exhaled breath condensate metabolites for predicting invasive lung adenocarcinoma included lysyl-valine, lysophosphatidylglycerol (19:0), vanillyl-glucose, hydroxyproline-threonine, phenylalanine, dihydroxybenzophenone, palmitoylglycine, triglyceride (43:0), monobutyl phthalate, hydroxyphenylpropionic acid, oleylglycine, dodecanedioic acid, p-chlorophenylalanine, and tauroursodeoxycholic acid.

[0007] A kit for predicting invasive lung adenocarcinoma comprises reagents required for detecting the contents of lysyl-valine, lysophosphatidylglycerol (19:0), vanillyl-glucose, hydroxyproline-threonine, phenylalanine, dihydroxybenzophenone, palmitoylglycine, triglyceride (43:0), monobutyl phthalate, hydroxyphenylpropionic acid, oleylglycine, dodecanedioic acid, p-chlorophenylalanine and tauroursodeoxycholic acid in an extract solution of an exhaled breath condensate sample.

[0008] Furthermore, the kit adopts liquid chromatography-mass spectrometry technology, and the reagents include methanol, acetonitrile and isopropanol.

[0009] A method for predicting invasive lung adenocarcinoma, comprising the following steps:

[0010] Step 1, taking an exhaled breath condensate sample of a subject to extract a solution, and detecting the content of lysyl-valine in the extract as a, the content of lysophosphatidylglycerol as b, the content of vanillyl-glucose as c, the content of hydroxyproline-threonine as d, the content of phenylalanine as e, the content of dihydroxybenzophenone as f, the content of palmitoylglycine as g, the content of triglyceride as h, the content of monobutyl phthalate as i, the content of hydroxyphenylpropionic acid as j, the content of oleylglycine as k, the content of dodecanedioic acid as l, the content of p-chlorophenylalanine as m, and the content of tauroursodeoxycholic acid adenosine phosphate as n, wherein the contents are all mass fractions;

[0011] Step 2: Calculate the probability of invasive lung adenocarcinoma Prob. If Prob < 0.5, the subject is judged to have invasive lung adenocarcinoma. The calculation method of the probability of invasive lung adenocarcinoma is: Prob = 1 / (1 + e -X ), where X = 10.695a + 1.182b - 42.394c - 3.919d + 176.644e - 1671.240f + 775.353g - 1470.690h + 985.416.

[0012] The beneficial effects of the present invention are as follows: the metabolites lysyl-valine, lysophosphatidylglycerol (19:0), vanillyl-glucose, hydroxyproline-threonine, phenylalanine, dihydroxybenzophenone, palmitoylglycine, triglyceride (43:0), monobutyl phthalate, hydroxyphenylpropionic acid, oleylglycine, dodecanedioic acid, p-chlorophenylalanine, and tauroursodeoxycholic acid in the exhaled breath condensate can be used in combination for the identification of patients with invasive lung adenocarcinoma. The combined markers of the present invention have the characteristics of high sensitivity and high specificity for the diagnosis of pleural diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of changes in the contents of lysyl-valine, lysophosphatidylglycerol (19:0), vanillyl-glucose, hydroxyproline-threonine, phenylalanine, dihydroxybenzophenone, palmitoylglycine, triglyceride (43:0), monobutyl phthalate, hydroxyphenylpropionic acid, oleylglycine, dodecanedioic acid, p-chlorophenylalanine and tauroursodeoxycholic acid in exhaled breath condensate of patients with invasive lung adenocarcinoma according to Examples 1 and 2 (expressed as mean ± standard error);

[0014] Figure 2 The ROC curve of the combined marker in distinguishing the invasive lung adenocarcinoma disease group. DETAILED DESCRIPTION

[0015] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0016] The 14 small molecule metabolites in the exhaled breath condensate determined by the present invention: lysyl-valine, lysophosphatidylglycerol (19:0), vanillyl-glucose, hydroxyproline-threonine, phenylalanine, dihydroxybenzophenone, palmitoylglycine, triglyceride (43:0), monobutyl phthalate, hydroxyphenylpropionic acid, oleylglycine, dodecanedioic acid, p-chlorophenylalanine, tauroursodeoxycholic acid are all important metabolites in the human body. The present invention combines the above 14 metabolites for the first time and applies them to the diagnosis of invasive lung adenocarcinoma. The mass spectrometry targeted analysis ion information of the combination is shown in Table 1 below.

[0017] Table 1. Information table of targeted analysis of 14 combination markers

[0018]

[0019] The detection method according to the kit is as follows:

[0020] (1) Exhaled breath condensate sample pretreatment method:

[0021] The exhaled breath condensate sample was thawed at 4°C, 100 μL of it was taken, and 400 μL of methanol extract containing multiple internal standards was added to precipitate the protein: vortexed for 30-90s, allowed to stand for 15-30mins, centrifuged at 10000-14000×g for 10-15mins at 4°C, and the supernatant was freeze-dried; 50uL of 10-20% (v / v) methanol aqueous solution was added, and the supernatant was centrifuged at 10000-14000×g for 10-15mins at 4°C, and the supernatant was analyzed by LC-MS / MS.

[0022] (2) Targeted detection of target metabolites using liquid chromatography-mass spectrometry:

[0023] The separation system is ultra-high performance liquid chromatography, the chromatographic column is C8, the mobile phase flow rate is 0.2-0.35ml / min, the column temperature is 40-60 degrees Celsius, and the injection volume is 5-10ul (C18 chromatographic column can also be used, the column temperature is 40-60 degrees Celsius, and the flow rate is 0.2-1.0ml / min); the eluent is an aqueous solution of 0.1% (v / v) formic acid in phase A, and an acetonitrile solution of 0.1% (v / v) formic acid in phase B (0-0.1% (v / v) formic acid aqueous solution and acetonitrile (methanol) solution eluent can also be selected, but the sensitivity is relatively lower); the internal standard is usually selected as an isotope, and other commonly used internal standards for mass spectrometry detection can also be used. The mass spectrometer used by the detector is a highly sensitive single quadrupole mass spectrometer or a tandem quadrupole mass spectrometer (Q-trap MS, QQQ MS), and the positive ion mode is detected. The mass spectrometry targeted analysis ion information of this combination is shown in Table 1.

[0024] (3) Judgment model based on mass spectrometry data:

[0025] The obtained data were analyzed by binary logistic regression using SPSS software, and the regression equation obtained by the constructed model is as follows:

[0026] Prob = 1 / (1+e -X )

[0027] in:

[0028] X=10.695a+1.182b-42.394c-3.919d+176.644e-1671.240f+775.353g-1470.690h+985.416;

[0030] The exhaled breath condensate sample of the subject was taken to extract the solution, and the content of lysyl-valine in the extract was detected to be a, the content of lysophosphatidylglycerol (19:0) was b, the content of vanillyl-glucose was c, the content of hydroxyproline-threonine was d, the content of phenylalanine was e, the content of dihydroxybenzophenone was f, the content of palmitoylglycine was g, the content of triglyceride (43:0) was h, the content of monobutyl phthalate was i, the content of hydroxyphenylpropionic acid was j, the content of oleylglycine was k, the content of dodecanedioic acid was l, the content of p-chlorophenylalanine was m, and the content of tauroursodeoxycholic acid adenosine phosphate was n.

[0031] An exhaled breath condensate sample is collected from the subject, metabolites are extracted, and the contents of lysyl-valine, lysophosphatidylglycerol (19:0), vanillyl-glucose, hydroxyproline-threonine, phenylalanine, dihydroxybenzophenone, palmitoylglycine, triglyceride (43:0), monobutyl phthalate, hydroxyphenylpropionic acid, oleylglycine, dodecanedioic acid, p-chlorophenylalanine, and tauroursodeoxycholic acid in the exhaled breath condensate sample are quantified by mass spectrometry. Prob is the probability of invasive lung adenocarcinoma, and Cutoff=0.5, that is, when the Prob value is less than 0.5, it is diagnosed as a benign pulmonary nodule.

[0032] The model has good discrimination ability for malignant pleurisy and invasive lung adenocarcinoma (see Figure 2 ), the AUC of the combined markers was 0.975, the sensitivity was 96.7%, and the specificity was 94.7%. This indicates that the 14 combined markers have the potential to diagnose invasive lung adenocarcinoma.

[0033] Example 1

[0034] Inclusion criteria for invasive lung adenocarcinoma: clinical manifestations of invasive lung adenocarcinoma: low fever, night sweats, fatigue, chest tightness, exudative pleural effusion, etc.; at the same time, the exhaled breath condensate and / or sputum culture is positive for Mycobacterium tuberculosis, and / or pleural tissue pathology examination is positive for tuberculosis; and those who are effective in subsequent anti-tuberculosis treatment are diagnosed as patients with invasive lung adenocarcinoma.

[0035] The inclusion criteria for the malignant pleurisy group were: exudative pleural effusion, tumor cells found in exfoliated cells of expired breath condensate, and / or pleural histopathological examination confirmed tumor pleural metastasis.

[0036] Exhaled breath condensate samples were used under the same conditions: 90 exhaled breath condensate samples from patients with malignant pleurisy and 30 exhaled breath condensate samples from patients with invasive lung adenocarcinoma; 50 ml of exhaled breath condensate was collected from patients before any treatment after admission, and the exhaled breath condensate was separated within half an hour and stored in a -80℃ refrigerator for examination.

[0037] 2. Analytical methods

[0038] 2.1 Pretreatment of exhaled breath condensate samples

[0039] The exhaled breath condensate sample was thawed at 4°C, 100 μL of it was taken, and 400 μL of methanol extract containing multiple internal standards was added to precipitate the protein: vortexed for 30-90s, allowed to stand for 15-30mins, centrifuged at 10000-14000×g for 10-15mins at 4°C, and the supernatant was freeze-dried; 50uL of 10-20% (v / v) methanol aqueous solution was added, and centrifuged at 12000×g for 15mins at 4°C, and the supernatant was taken for LC-MS / MS analysis, and the peak areas of the internal standard peak and the eight metabolites were calculated by the extracted ion flow chart to determine the contents of the eight metabolites. The internal standards and their contents in the extractant are shown in Appendix 1.

[0040] 2.2 Ultra-high performance liquid chromatography-mass spectrometry analysis

[0041] (1) Liquid phase 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 (1.7 um, 2.1 mm × 100 mm) (Waters, Ireland); the mobile phase A was a mobile phase: phase A was an aqueous solution containing 0.1% (v / v) formic acid, and phase B was a 0.1% (v / v) formic acid acetonitrile solution; the elution gradient was: 2% phase B from 0 to 1 min, linearly changed to 80% phase B from 1 min to 20 min, linearly changed to 100% phase B from 20 min to 25 min, the phase was maintained for 4 min, and linearly decreased to 2% phase B within 0.1 min and maintained for 1 min; the column temperature was 50 °C; the mobile phase flow rate was 0.35 mL / min; the injection volume was 10 μL.

[0042] (2) Mass spectrometry conditions: The mass spectrometer was a quadrupole ion trap mass spectrometer (Q-Trap 5500MS) (AB SCI EX, Framingham, MA, USA); positive ion mode detection was adopted; the curtain gases, GAS1, and GAS2 were set to 50 arb itrary units, 35 arb itrary units, and 35 arb itrary units, respectively; the ion source temperature was 500°C, and the spray voltage was 5.0 kV.

[0043] 2.3 Exhaled breath condensate test results and auxiliary diagnosis methods

[0044] For the malignant pleurisy group, lysyl-valine, lysophosphatidylglycerol (19:0), vanillyl-glucose, hydroxyproline-threonine, phenylalanine, dihydroxybenzophenone, palmitoylglycine, triglyceride (43:0), monobutyl phthalate, hydroxyphenylpropionic acid, oleylglycine, dodecanedioic acid, p-chlorophenylalanine, and tauroursodeoxycholic acid were significantly changed (see Figure 1 ). At the same time, the content of each marker was substituted into the regression equation to calculate the probability. The cutoff value used was 0.5, that is, if the probability of the combined marker was less than 0.5, it was considered to be a benign pulmonary nodule. The AUC of the combined marker was 0.973, and the sensitivity and specificity were also relatively high, at 95.5% and 94.3%, respectively (see Figure 2 ).

[0045] Example 2

[0046] In order to verify the stability and reliability of the combined markers, the sample collection source was changed and external independent samples were used for verification. The analytical methods, instruments and reagents used in this experiment were completely consistent with those in Example 1.

[0047] Inclusion criteria for invasive lung adenocarcinoma: clinical manifestations of invasive lung adenocarcinoma: low fever, night sweats, fatigue, chest tightness, exudative pleural effusion, etc.; at the same time, the exhaled breath condensate and / or sputum culture is positive for Mycobacterium tuberculosis, and / or pleural tissue pathology examination is positive for tuberculosis; and those who are effective in subsequent anti-tuberculosis treatment are diagnosed as patients with invasive lung adenocarcinoma.

[0048] The inclusion criteria for the malignant pleurisy group were: exudative pleural effusion, tumor cells found in exfoliated cells of expired breath condensate, and / or pleural histopathological examination confirmed tumor pleural metastasis.

[0049] Exhaled breath condensate samples were used under the same conditions: exhaled breath condensate samples from 50 patients with malignant pleurisy and 15 patients with invasive lung adenocarcinoma; 50 ml of exhaled breath condensate was collected from the patients before any treatment after admission, and the exhaled breath condensate was separated within half an hour and stored in a -80°C refrigerator for examination.

[0050] 2. Exhaled breath condensate test results and auxiliary diagnosis methods

[0051] For the malignant pleurisy group, lysyl-valine, lysophosphatidylglycerol (19:0), vanillyl-glucose, hydroxyproline-threonine, phenylalanine, dihydroxybenzophenone, palmitoylglycine, triglyceride (43:0), monobutyl phthalate, hydroxyphenylpropionic acid, oleylglycine, dodecanedioic acid, p-chlorophenylalanine, and tauroursodeoxycholic acid were significantly changed (see Figure 1). At the same time, the content of each marker was substituted into the regression equation to calculate the probability. The cutoff value used was 0.5, that is, if the probability of the combined marker was less than 0.5, it was considered to be a benign pulmonary nodule. The AUC of the combined marker was 0.941, and the sensitivity and specificity were also relatively high, at 85.0% and 94.1%, respectively (see Figure 2 ).

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A combination of exhaled breath condensate metabolites for predicting invasive lung adenocarcinoma, characterized in that: These include lysyl-valine, lysophosphatidylglycerol, vanillyl-glucose, hydroxyproline-threonine, phenylalanine, dihydroxybenzophenone, palmitoylglycine, triglycerides, monobutyl phthalate, hydroxyphenylpropionic acid, oleylglycine, dodecanedioic acid, p-chlorophenylalanine, and tauroursodeoxycholic acid.

2. A kit for predicting invasive lung adenocarcinoma, characterized in that: The invention comprises reagents required for detecting the contents of lysyl-valine, lysophosphatidylglycerol, vanillyl-glucose, hydroxyproline-threonine, phenylalanine, dihydroxybenzophenone, palmitoylglycine, triglyceride, monobutyl phthalate, hydroxyphenylpropionic acid, oleylglycine, dodecanedioic acid, p-chlorophenylalanine and tauroursodeoxycholic acid in the extraction solution of the exhaled breath condensate sample.

3. The kit for predicting invasive lung adenocarcinoma according to claim 2, characterized in that: The method adopts liquid chromatography-mass spectrometry technology, and the reagents include methanol, acetonitrile and isopropanol.

4. A method for predicting invasive lung adenocarcinoma, characterized in that: The following steps are involved: Step 1, taking an exhaled breath condensate sample of a subject to extract a solution, and detecting the content of lysyl-valine in the extract as a, the content of lysophosphatidylglycerol as b, the content of vanillyl-glucose as c, the content of hydroxyproline-threonine as d, the content of phenylalanine as e, the content of dihydroxybenzophenone as f, the content of palmitoylglycine as g, the content of triglyceride as h, the content of monobutyl phthalate as i, the content of hydroxyphenylpropionic acid as j, the content of oleylglycine as k, the content of dodecanedioic acid as l, the content of p-chlorophenylalanine as m, and the content of tauroursodeoxycholic acid adenosine phosphate as n, wherein the contents are all mass fractions; Step 2: Calculate the probability of invasive lung adenocarcinoma Prob. If Prob < 0.5, the subject is judged to have invasive lung adenocarcinoma. The calculation method of the probability of invasive lung adenocarcinoma is: Prob = 1 / (1 + e -X ), where X = 10.695a + 1.182b - 42.394c - 3.919d + 176.644e - 1671.240f + 775.353g - 1470.690h + 985.416.

Images