CN111796033A - Lung cancer gas markers in exhaled breath and their application in lung cancer screening - Google Patents

Abstract

The invention discloses a biomarker for screening early lung cancer, wherein the biomarker is acetaldehyde in human expiration. According to the invention, the content of acetaldehyde in expiration of a lung cancer patient is found to be reduced for the first time, and meanwhile, the acetaldehyde released by lung cancer cells is far lower than that of normal lung epithelial cells through in vitro cell further verification, so that the acetaldehyde can be used as a biomarker to be applied to lung cancer screening.

Description

Lung cancer gas markers in exhaled breath and their application in lung cancer screening

technical field

The invention relates to the technical field of lung cancer detection, in particular to the application of acetaldehyde in exhaled breath in rapid non-invasive screening of lung cancer.

Background technique

Lung cancer is the most diagnosed type of cancer globally each year, and it also causes the most deaths of all cancers each year. The survey shows that the five-year survival rate of lung cancer patients will drop from 45% in stage I to 1% in stage IV. However, because there are no obvious symptoms in the early stage of lung cancer, most patients are already in stage IV when they are discovered, resulting in a low overall survival rate. . Therefore, there is an urgent need for a lung cancer screening technique. The commonly used screening method in clinical practice is low-dose computed tomography (LDCT), but this technique still cannot identify early pulmonary nodules as malignant or benign. Moreover, the technology is expensive and not suitable for large-area screening.

Breath detection technology has been proved by a large number of studies to be a potential lung cancer screening method, but a unified and mutually verifiable lung cancer breath marker has not yet been found. The markers have not been validated by multicenter etc. Therefore, there is an urgent need to develop a standardized and verifiable lung cancer marker to solve the problem of early diagnosis of lung cancer.

SUMMARY OF THE INVENTION

In order to make up for the deficiency of the existing lung cancer early screening technology, the present invention provides a biomarker acetaldehyde for early lung cancer screening and its rapid non-invasive screening for lung cancer by conducting a multi-center study of exhalation environment monitoring and condition standardization. applications in .

In a first aspect, the present invention provides a biomarker for lung cancer screening in human breath, characterized in that the biomarker is gaseous acetaldehyde.

In a second aspect, the present invention provides a method for screening lung cancer patients, the method comprising the steps of:

1) Collect breath samples from subjects, including patients with suspected lung cancer and normal people;

2) Detecting the breath samples collected in step 1);

3) The acetaldehyde content in the exhaled breath of normal people and suspected lung cancer patients was compared and analyzed, and the suspected lung cancer patients were determined to be positive or negative.

In a specific embodiment, subjects fasted after 20:00 the night before breath sample collection, sat for 5 minutes before breath sample collection, gargled with cool purified water, and then performed air blowing and sample collection.

In a specific embodiment, preferably, the breath samples collected in step 1) are detected by sensors and/or chromatography and/or mass spectrometry and/or spectroscopic techniques, more preferably, a mass spectrometer is used for detection; further preferably, The mass spectrometer used was a proton transfer reaction mass spectrometer.

In a specific embodiment, the mass scanning range of the mass spectrometer is set to m/z20-m/z150, the temperature of the reaction tube is 20°C, and the gas pressure is 1.46 Torr.

In a third aspect, the present invention provides a use of a lung cancer biomarker in the preparation of a reagent for lung cancer screening, wherein the biomarker is gaseous acetaldehyde.

In a fourth aspect, the present invention provides a detection system for detecting lung cancer biomarkers, which is characterized by comprising a gas collection device, detection equipment and report generation software.

In a specific embodiment, the gas collection device adopts two modes: online and offline, wherein the online mode directly connects the mouth and the detection equipment through a polytetrafluoro pipe to realize online real-time monitoring; the offline mode uses a glass material sampling bottle to collect Subjects exhale and take precautions against light.

Further, the detection device includes a sensor or a chromatographic or mass spectrometer or a spectral signal detection device; the report generation software gives the subject information, including the acetaldehyde concentration in the exhaled breath and the lung cancer positive or negative judgment result.

Description of drawings

Figure 1 (left) is an arbitrary pair of exhaled mass spectra of lung cancer patients and normal people, and Figure 1 (right) is the statistical result of acetaldehyde content in the exhaled breath of all lung cancer patients and normal people.

Figure 2 shows the content of acetaldehyde released by lung epithelial cells and adenocarcinoma and squamous cell carcinoma cells.

Detailed ways

After extensive and in-depth research, the present invention uses mass spectrometry technology to compare the gas composition and concentration changes in the exhaled breath of lung cancer patients and healthy people. Center validation, as well as in vitro cell culture validation, demonstrate acetaldehyde as a biomarker for early lung cancer screening.

Biomarkers

A "biomarker" refers to a molecular indicator with a specific biological property, biochemical signature, that can be used to determine the presence or absence and/or severity of a particular disease or condition.

In the present invention, "biomarker" refers to a molecular compound produced by human metabolism, namely acetaldehyde, which indicates the presence or absence of a specific disease lung cancer by measuring its concentration.

Detection technology (method)

"Detecting the acetaldehyde content in the breath" refers to determining the presence of acetaldehyde in the breath and its volume ratio to the total substance, so as to predict lung cancer. The detection of acetaldehyde in the present invention can be achieved by a variety of detection technologies that already exist in the art , these techniques include but are not limited to sensors, chromatography, mass spectrometry, spectroscopic techniques.

The sensor described in the present invention can ionize the measured substance by means of photoionization or flame ionization, and then amplify and convert it into an electrical signal output by an electron multiplier to obtain the concentration information of the measured substance.

The chromatography in the present invention first separates the tested substance through a chromatographic column, then uses photoionization or flame ionization to ionize the tested substance, and then amplifies and converts it into an electrical signal output by an electron multiplier to obtain the measured substance. concentration information.

The mass spectrometry technique described in the present invention ionizes the substance to be tested by means of chemical ionization, electron bombardment ionization, electrospray ionization, etc., and identifies its molecular weight, which is amplified and converted into an electrical signal output by an electron multiplier to obtain the concentration information of the substance to be tested.

In the present invention, the spectrometer uses a photodetector such as a photomultiplier tube to measure the intensity of the spectral line at different wavelength positions to determine the type and content of the substance.

Lung Cancer Screening Products

The invention takes breath acetaldehyde as the detection object, and invents a product that can be used for non-invasive screening of lung cancer. In addition to the above sensors, chromatography, mass spectrometry, and spectral signal detection equipment, it should also include a gas collection device and report generation software.

The gas collection device adopts two methods: online and offline:

The online method directly connects the mouth and the detection instrument through a PTFE pipe to realize online real-time monitoring and avoid changes in the content of acetaldehyde during sample preservation.

In offline mode, glass sampling bottles were used to collect the breath of the subjects, and measures to avoid light were taken to avoid changes in the content of acetaldehyde caused by photolysis.

The report generation software gives subject information, including name, gender, age, contact information; as well as acetaldehyde concentration in exhaled breath and lung cancer positive and negative.

The experiments in the present invention are completed at least three times, and the result data are all expressed in the form of mean ± standard deviation. SPSS22.0 statistical software is used for statistical analysis. The difference between lung cancer patients and healthy people, lung cancer patients Cells were compared with normal lung epithelial cells using the u test and considered statistically significant when p≤0.05.

The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention, and are not meant to limit the protection scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 Screening of breath biomarkers associated with lung cancer

(1) Collection of subjects' breath samples

The present invention screens out the early-stage lung cancer biomarkers in the exhalation by analyzing the exhalation of early-stage lung cancer patients and normal people. Breath samples were obtained from three experimental centers: 22 lung cancer patients and 90 normal subjects in the first experimental center, 27 lung cancer patients and 88 normal subjects in the second experimental center, and 29 lung cancer patients and 111 normal subjects in the third experimental center. Normal people, lung cancer patients in the diagnosis process using tissue biopsy, computed tomography (CT) scan or puncture three methods. All normal controls were accompanying family members in the same ward as the patients. Informed consent of the patients was obtained for the breath experiments, and all were approved by the organizational ethics committee. The exhalation sample collection adopts the method of online insufflation, and the subject only needs to exhale for 8s at the sampling port of the proton transfer reaction mass spectrometer to complete the sample collection. Fasting after 20:00 the night before breath sample collection, including beverages other than pure water. Before the breath sample collection, sit still for 5 minutes, rinse your mouth with cold purified water, and start blowing for breath sample collection.

(2) Breath detection

Breath detection is done by mass spectrometry, and the mass spectrometer used is a proton transfer reaction mass spectrometer (Ion Sniffer2020Q), which is an online gas sample detection instrument. The mass spectrometer sucks the sample inside, completing the detection. The mass scan range was set to m/z20-m/z150, the reaction tube temperature was 20°C, and the gas pressure was 1.46 Torr.

(3) Results

SPSS22.0 statistical software was used for statistical analysis. The results are shown in Figure 1. The content of acetaldehyde in the exhaled breath of lung cancer patients was significantly lower than that in the exhaled breath of healthy people, suggesting that acetaldehyde can be used as a biomarker for lung cancer.

Example 2 Comparison of acetaldehyde released by lung cancer cells and normal lung epithelial cells

(1) Cell culture

The common lung cancer cell squamous cell carcinoma cell CALU-1, adenocarcinoma cell SPC-A1 and normal lung epithelial cell BEAS-2B were selected as the detection objects. The petri dish model is RPMI-1640, add 10% fetal bovine serum and 100U/mL double antibody, and culture at 37 °C and 5% CO _2. After the cells grow well, continue to culture for 24 hours after changing the medium. for detection.

(2) Testing instruments

The detection instrument is gas chromatography-mass spectrometry, the column model is TG-624SiIMS (30m×0.32mm×1.8μm), the heating program is 50°C for 5min, and then rises to 180°C at a heating rate of 15°C/min and maintains 1min. The inlet, transfer line and ion source temperatures were all 200°C.

(3) Sampling method

Cell culture dishes were enriched for headspace gas using 75 μm Carboxen/PDMS solid phase microextraction (SPME) for 40 min.

(4) Results

As shown in Figure 2, there are significant differences in the release of acetaldehyde from lung cancer cells and normal lung epithelial cells, and the acetaldehyde release of lung cancer cells is significantly lower than that of normal epithelial cells, which further proves that acetaldehyde can be used as a biomarker of lung cancer cells.

The parts of the present specification that are not explained in detail belong to the well-known technology in the art.

The above embodiments are provided for the purpose of describing the present invention only, and are not intended to limit the scope of the present invention. The scope of the invention is defined by the appended claims. Various equivalent replacements and modifications made without departing from the spirit and principle of the present invention should be included within the scope of the present invention.

Claims (9)

1. A biomarker for lung cancer screening in human exhalation, wherein the biomarker is gaseous acetaldehyde. 2. a method for detecting lung cancer patient, it is characterised in that the method comprises the following steps: 1) Collect breath samples from subjects, including patients with suspected lung cancer and normal people; 2) Detecting the breath samples collected in step 1); 3) The acetaldehyde content in the exhaled breath of normal people and suspected lung cancer patients was compared and analyzed, and the suspected lung cancer patients were determined to be positive or negative. 3. The method according to claim 2, wherein the subject fasts after 20:00 the night before the breath sample collection, sits for 5 minutes before the breath sample collection, and rinses the mouth with cold purified water. Perform air blow and sample collection. 4. The method according to claim 2 or 3, wherein the breath samples collected in step 1) are detected by sensors and/or chromatographic and/or mass spectrometry and/or spectroscopic techniques, preferably, a mass spectrometer is used detection; further preferably, the mass spectrometer used is a proton transfer reaction mass spectrometer. 5 . The method according to claim 4 , wherein the mass scanning range during detection by the mass spectrometer is set to m/z20-m/z150, the temperature of the reaction tube is 20°C, and the air pressure is 1.46 Torr. 6 . 6. Use of a lung cancer biomarker in preparing a reagent for lung cancer screening, wherein the biomarker is gaseous acetaldehyde. 7. A detection system for detecting lung cancer biomarkers, characterized by comprising a gas collection device, detection equipment and report generation software. 8. The detection system according to claim 7, characterized in that, the gas collection device adopts two modes, online and offline, wherein the online mode directly connects the mouth and the detection equipment through a polytetrafluoro pipe, so as to realize online real-time Monitoring; off-line use glass sampling bottles to collect subjects' breath, and take measures to protect from light. 9. The detection system according to claim 7 or 8, wherein the detection device comprises a sensor or a chromatographic or mass spectrometer or a spectral signal detection device; the report generation software provides subject information, including Acetaldehyde concentration and lung cancer positive or negative judgment results.

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