Lung cancer is the malignant tumor with the highest incidence and death rate in the world, and in the past 20 years, lung cancer has caused more than 20% of the total deaths due to cancer in China. Because the clinical symptoms of early lung cancer are not obvious, 80% of lung cancer patients are already in the middle and late stages when they are diagnosed, and more than 50% of them are inoperable and the 5-year survival rate is only 10%. The key to improve the prognosis lies in early detection and early diagnosis. Exhaled breath condensate (EBC) has the advantages of being non-invasive, easy to perform and repeatable, and the significant changes of some components can reflect the pathophysiological status of the lung and airway. However, it is still in the exploration stage and there are still many unsolved problems. Lu Yanda, Department of Radiotherapy, Affiliated Hospital of Hainan Medical College
1 Overview of EBC detection
There are three types of devices for EBC collection: the RTUBE portable device produced in the United States, which has a polypropylene tube in the middle, and the condensed liquid can be attached to the inner tube wall; the Ecoscreen electric cooling device produced by Ehrlich Jaeger GmbH in Germany; and there are also homemade condensing devices in China. The report. All include a refrigeration tube with a one-way valve, which can separate the inhaled and exhaled air to prevent repeated inhalation of exhaled air, and the collection requires the subject to rinse the mouth, with a nasal clip to exclude the nasal cavity, nasal security generated by the interference of substances, saliva when asked to swallow, coughing when the collection needs to be suspended. The subject breathes through the mouth for 10-15 min, and 1-3 ml of condensate can be collected in the condenser. The specimen collected needs to be tested for amylase content to determine whether there is contamination by saliva. Compared with traditional testing methods, EBC testing has the advantages of being truly non-invasive, instantaneous, allowing dynamic monitoring, and being applicable to a broader population.
EBC is a highly dilute body fluid, and a special, highly sensitive assay can measure volatile substances (such as nitric oxide, carbon monoxide, etc.), non-volatile substances (such as nitrite, ammonia and vasoactive amines), proteins, peptides, nucleic acids and some lipid metabolites. Studies have reported that thousands of substances can be detected in EBCs, and new substances are constantly being detected, mainly from the lower airway lining fluid, presumably due to “cyclones” formed by the respiratory airflow that “scrape” the airway lining fluid off or the expansion of the airway during inspiration. The tension created by inspiration “tears” the superficial fluid phase. These substances can change with disease and therapeutic interventions. Changes in these biochemical molecules (generally less than 65,000 relative molecular masses) or in parameters such as pH in EBCs can reflect oxidative damage, inflammation, and other airway states.
Research on EBC testing has mainly focused on the study of detectable factors and pH in EBC using case-control studies or cross-sectional and longitudinal studies to provide an experimental basis for clinical diagnosis and differential diagnosis, and on the search for possible factors affecting EBC testing, such as collection equipment, collection methods and testing methods, to conduct research on standardization of EBC testing. At present, there are more studies on inflammatory markers in EBC at home and abroad, such as hydrogen peroxide, nitric oxide and its related products (nitrate/nitrite), prostaglandins, leukotrienes, etc., which have important applications in the I monitoring and treatment of airway inflammation. Many scholars have found through research that finding lung cancer-specific tumor markers in EBC and combining multiple markers for lung cancer diagnosis also have important applications for lung cancer screening, early diagnosis, disease monitoring and prognosis determination.
2 Main detectable factors in EBC of lung cancer patients and their significance
The formation of lung cancer is a multi-stage, multi-step and multi-gene process, in which different genes or cytokines are changed at different stages one after another or at the same time. Currently, there are more and more studies on the detection of lung cancer markers in EBC, which are mainly divided into specific and non-specific markers, the former including oxidative stress or inflammatory markers, and the latter mainly including markers related to tumor angiogenesis and gene mutations, etc.
In 1994, Khyshiktyev et al. suggested that the level of lipid peroxidation products in EBC of lung cancer patients was lower than that of normal population. were lower than those of the normal population. Later, a study showed that serum levels of 8-isoprostane were increased in lung cancer patients with healthy smokers as controls and were more significantly elevated in progressive lung cancer, while their levels were not elevated in EBC of lung cancer patients and were not significantly associated with clinical stage and age. Another scholar examined the EBC of 21 non-small cell lung cancer patients, 21 healthy non-smokers, 13 former smokers, and 16 current smokers, and found that the levels of 8-iso-prostaglandins and hydrogen peroxide were significantly higher in the EBC of lung cancer patients than in the smoking population. The level of lipid peroxidation products in EBC of lung cancer patients is still controversial, and oxidative stress is closely related to many diseases, so its quantitative index needs to be further explored in the application of early diagnosis and disease monitoring of lung cancer.
2.2 Growth factor/endothelin Endothelin-1 is a peptide consisting of 21 amino acids, which has the ability to regulate mitosis and vasodilation. Carpagnano et al. found that elevated levels of endothelin-1 could be detected in most non-small cell lung cancer cell lines, and examined the levels of endothelin-1 in EBCs of 30 lung cancer patients, compared with those of healthy controls. Compared with healthy controls, its level was significantly higher and positively correlated with lung cancer stage, and the level of endothelin-1 in EBC decreased accordingly in stage I lung cancer patients after surgery, leading to the preliminary conclusion that endothelin-1 in EBC can be used as a marker for early diagnosis of lung cancer and can also be applied for disease monitoring of lung cancer.
In addition, Carpagnano et al. found that the levels of interleukin 2 and interleukin 6 were significantly increased in EBC of patients with non-small cell lung cancer, but the specificity of these inflammatory cytokines was not high and could be accompanied by a variety of lesions such as trauma, inflammation and tumors in the body. It has been proposed that increased levels of endothelin-1 can also be seen in EBCs of patients with pulmonary hypertension, pulmonary fibrosis and inflammatory lung diseases; levels of interleukin 6 are significantly increased in EBCs of patients with chronic obstructive pulmonary disease and asthma, so these growth factor-like markers have limitations when applied to lung cancer alone. When inconsistent results occur, they should be analyzed and studied in combination with other lung cancer markers, thus providing more experimental basis for their application in early diagnosis of lung cancer and other aspects.
2.3 Angiogenesis-related factors Studies have confirmed that various active substances such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are involved in regulating tumor angiogenesis and can be used as tumor-specific molecular markers. The levels of VEGF, bFGF, and angiopoietin were significantly higher in EBCs of patients with non-small cell lung cancer compared with those of healthy subjects and patients with chronic obstructive pulmonary disease, but were not significantly associated with lung cancer stage, anatomy, or histological type. The levels of vascular-related factors in EBCs decreased significantly in patients after 2 chemotherapy sessions (at least 25% reduction in mass diameter). It is tentatively believed that the detection of the levels of vascular-related factors in EBCs is important for guiding lung cancer treatment and monitoring treatment response, but large-scale basic and clinical studies are still needed to provide experimental basis.
2.4 Nucleic acids in the process of lung carcinogenesis and development, a large amount of DNA from cancerous bronchial or airway epithelial cells can be shed directly into the lining fluid of the airways, and it is presumed that gene expression analysis in EBC for early diagnosis of lung cancer is feasible. The current hot spots mainly focus on probing specific microsatellite alteration sites, detecting p53 gene mutation and gene promoter methylation status in EBC.
2.4.1 Microsatellite alterations Hung et al. found frequent allele-specific loss at 3p14, 3p21 and 3p25 in lung cancer patients, and Miozzo et al. applied PCR combined with microsatellite analysis to find microsatellite instability at 3p21, 3p24 and 3p25. In 2005, Carpagnano et al. collected EBCs and whole blood from 30 patients with non-small cell lung cancer and 20 healthy subjects, extracted DNA and examined five microsatellite loci in the 3p region (D3S238, D3S1266, D3S1300, D3S1304, D3S1289). D3S1289) and found that microsatellite alterations were detectable in 53% of EBC-DNA and 10% of whole blood-DNA in the non-small cell lung cancer group, compared to 13% of EBC-DNA and 2% of whole blood-I DNA in healthy controls, indicating a higher sensitivity of EBC-DNA in probing for microsatellite alterations specific to lung cancer, in addition to the results showing a higher sensitivity of EBC-DNA in In addition, the results showed that the number of microsatellite alterations in EBC-DNA of lung cancer patients was directly and significantly correlated with the amount of smoking in the subjects, suggesting that this tool could be valuable in screening and diagnosis of lung cancer in high-risk groups.
Subsequently, Carpagnano et al. demonstrated that the results of microsatellite locus analysis in the 3p region of EBC-DNA of non-small cell lung cancer patients were consistent with those in the corresponding lung cancer tissues, and that the detection of EBC-DNA could reflect somatic cell mutations. In addition, it was found that the number of microsatellite alterations in the 3p region of EBC was significantly correlated with the prognosis of patients with non-small cell lung cancer, with heterozygote loss at the D3S1289 locus negatively correlated with the prognosis of patients with lung adenocarcinoma and heterozygote loss at the D3S2338 locus affecting the prognosis of patients with squamous lung cancer. It has been shown that ERCC, a rate-limiting enzyme of the nucleoside excision repair pathway, is also involved in lung carcinogenesis and development. Based on this, Carpagnano et al. studied 34 patients with non-small cell lung cancer, 33 healthy volunteers (19 nonsmokers and 14 smokers), detecting 2 microsatellite loci in the 19q region of DNA from EBC and whole blood: D19S908 (ERCC-1 locus) and D19S393 (ERCC-2 locus), and of which The results showed that microsatellite alterations were not detected in EBC and whole blood DNA of healthy non-smokers; microsatellite alterations were detectable in 16% and 25% of EBC-DNA of the healthy smoking group and non-small cell lung cancer group, respectively; the rate of microsatellite alterations in EBC-DNA was associated with the amount of smoking in subjects and patient prognosis (D19S393 heterozygote deletion was strongly associated with prognosis ), independent of lung cancer tissue type.
Although the number of samples examined was small and the study is still at an early stage, further in-depth studies are needed, but the researchers concluded that microsatellite alterations can be detected in EBC of lung cancer patients, and their positive rate is significantly higher than that of healthy controls, and the positive detection rate is higher than that of whole blood, which is feasible for early screening, diagnosis and disease monitoring and follow-up of lung cancer.
2.4.2 Mutant p53 gene Numerous investigations have shown that the presence of hotspot mutant codons in the p53 gene is associated with polycyclic aromatic hydrocarbon carcinogens (such as benzo(a)pyrene) in tobacco, and detection of p53 gene mutations can reflect the degree of damage of precancerous lesions, suggesting that detection of p53 gene mutation sites in EBC is specific for screening early lung cancer in high-risk groups. 2004 Gessner et al. recruited 18 patients with non-small cell lung cancer (all smokers) and 18 healthy volunteers (4 had quit smoking for more than 5 years and the rest were non-smokers), collected EBCs, extracted DNA, amplified exon 5 to exon 8 of the p53 gene using a nested PCR assay and sequenced them for observation, and found that four of the 11 patients with non-small cell lung cancer had p53 gene mutations (36.4%), while no p53 mutation was detected in healthy volunteers; and the p53 mutation detected in EBC was found to be inconsistent with the mutation detected in the corresponding tissues, which the investigators believe can be explained by regional carcinogenesis, as EBC can reflect the overall situation of airways and lung parenchyma, while biopsy can only reflect local carcinogenesis due to the limitation of sampling. It was also found that mutated p53 gene could be detected in EBC of patients with bronchial atypical hyperplasia. p53 protein-positive atypical hyperplastic cells with concurrent p53 gene mutation tend to maintain their irreversible atypical hyperplasia and partly develop into squamous carcinoma. Therefore, it is speculated that detection of p53 gene in EBC to monitor lung cancer is possible, but further research is needed and application to clinical use is still a gap.
2.4.3 DNA methylation has been experimentally demonstrated that promoter hypermethylation can directly or indirectly block transcription; it can also induce the conversion of cytosine to thymine through the deamination of 5-methylcytosine, leading to gene replacement mutations and abnormal gene expression; it can also cause structural changes of chromatin in the corresponding regions of the genome, resulting in highly helical chromatin and condensation into clusters, leading to unstable gene expression, etc. and thus It can also cause changes in chromatin structure in the corresponding regions of the genome, resulting in highly helical chromatin and condensed clusters, leading to destabilization of gene expression and thus inducing genetic diseases or tumors.
In 2009, Han et al. studied 17 lung cancer patients and 37 healthy volunteers, and examined the promoter methylation of Ras-related region family 1A gene (RASSF1A) and death-associated protein kinase gene (DAPK) in EBC using bisulfite treatment followed by sequencing. The results showed that the degree of promoter methylation and spatial distribution of the 2 genes had great individual differences; the degree of RASSF1A methylation correlated with the degree of smoking in the subjects; the CpG site in the -63 region of the DAPK gene differed between the lung cancer and non-lung cancer groups; and the degree of promoter methylation did not correlate with lung cancer stage and histology. These suggest that detection of gene methylation in EBC is feasible and has the potential to provide information about smoking exposure and disease status, etc.
3 Problems and outlook
The EBC test is a new method that provides early lesion signals in the lower respiratory tract and lung parenchyma, and its collection device is portable, collection is not affected by age, gender, or degree of disease, and the collection process does not change the respiratory environment, which makes the EBC test completely noninvasive, easy and fast, and enables real-time dynamic monitoring. It can be used in almost all patients (including pediatric patients, mechanically ventilated patients and comatose patients). The specific anatomical location of the source of the substances detected in EBC is unknown and lacks specificity; EBC is a highly diluted body fluid, and the concentration of some substances is extremely low, which is not easy to detect; the dilution of EBC varies greatly among individuals, and there is no uniform method to determine the dilution of EBC. The dilution of EBC varies greatly among individuals, and there is no uniform method to determine the dilution of EBC, which may affect the evaluation of the test results; no large-scale population studies have been reported, and there is a lack of normal reference values for various substances in EBC; different equipment or collection and testing methods used by investigators may yield different results, which also makes the results lack comparability; the reproducibility and stability of the method also need further studies to confirm. In addition, the specificity and sensitivity of this method for lung cancer diagnosis still need further experimental studies, however, with the further improvement of the detection technology and the continuous research on the process and molecular mechanism of lung cancer development, the detection of lung cancer-related biochemical markers in EBC will definitely show a significant role in clinical research on lung cancer pathogenesis, lung cancer screening, disease monitoring, efficacy assessment and follow-up. The results of this study will be presented in the following table.
2013-02-24 16:09 Source: International Journal of Respiratory Medicine Author: Liangliang Dong et al.