Plasma and EBC microRNAs as early biomarkers of non-small-cell lung cancer.

Lung cancer is a major cause of death in Western countries. Current screening methods are invasive and still lead to a high percentage of false positives. There is, therefore, a need to find biomarkers that increase the probability of detecting lung cancer early. MicroRNAs (miRNAs) are stable molecules in blood plasma and exhaled breath condensate (EBC). We quantified miRNA-21 and miRNA-486 expression from plasma and EBC samples from patients with a diagnosis of non-small-cell lung cancer (NSCLC) and controls. miRNA-21 was significantly higher in plasma and in EBC of the NSCLC patients and miRNA-486 was significantly lower. This difference indicates a significantly improved diagnostic value, and suggests that these miRNAs could be clinically used as a first-line screening test in high-risk subjects.

Inter- and intra-subject variability of kinetics of airway exhalation and deposition of particulate matter in indoor polluted environments.

PM(2.5) generated by indoor combustion activities can contribute significantly to personal PM exposure. The aims of this study were: (1) to validate a device specifically designed to study the kinetics of particle exhalation and the percentage of airway particle deposition (%DEP) in polluted indoor environments (welding fumes, environmental tobacco smoke - ETS) and (2) to assess the intra- and inter-subject variability of the signal. The device was tested on 14 subjects exposed to welding fumes and 10 subjects exposed to environmental tobacco smoke (ETS), performing repeated measures at different environmental PM concentrations. The intra-subject variability of the signal for particles with diameter 0.3-1.0 µm showed a geometric mean of %CV always below 6%, despite the values of %DEP. In the welding fume study, the increase in airborne 0.5-1.0 µm PM concentrations between the consulting room and production department was explainable in terms of increased density due to the metallic composition of particles. The %DEP of 0.3-1.0 µm ETS particles decreased with airborne PM concentration due to the technical limits of a laser particle counter and the perturbation induced by the physical characteristics of ETS PM. However, also at those extreme conditions, the signal remained repeatable and the individual susceptibility to PM remained substantially unaltered. In conclusion, the versatility and portability of our device, together with the repeatability of the signal, confirmed that the kinetics of exhaled particles and %DEP could be routinely measured in polluted environments and used to define individual susceptibility to airborne particles.

Expression levels of some antioxidant and epidermal growth factor receptor genes in patients with early-stage non-small cell lung cancer.

THIS STUDY WAS AIMED AT: (i) investigating the expression profiles of some antioxidant and epidermal growth factor receptor genes in cancerous and unaffected tissues of patients undergoing lung resection for non-small cell lung cancer (NSCLC) (cross-sectional phase), (ii) evaluating if gene expression levels at the time of surgery may be associated to patients' survival (prospective phase). Antioxidant genes included heme oxygenase 1 (HO-1), superoxide dismutase-1 (SOD-1), and -2 (SOD-2), whereas epidermal growth factor receptor genes consisted of epidermal growth factor receptor (EGFR) and v-erb-b2 erythroblastic leukaemia viral oncogene homolog 2 (HER-2). Twenty-eight couples of lung biopsies were obtained and gene transcripts were quantified by Real Time RT-PCR. The average follow-up of patients lasted about 60 months. In the cancerous tissues, antioxidant genes were significantly hypo-expressed than in unaffected tissues. The HER-2 transcript levels prevailed in adenocarcinomas, whereas EGFR in squamocellular carcinomas. Patients overexpressing HER-2 in the cancerous tissues showed significantly lower 5-year survival than the others.

Chromium in exhaled breath condensate (EBC), erythrocytes, plasma and urine in the biomonitoring of chrome-plating workers exposed to soluble Cr(VI).

Chromium (Cr) levels measured in exhaled breath condensate (EBC-Cr) and urine (Cr-U) at the beginning and end of working shifts were related to those measured in erythrocytes (Cr-RBC) and plasma in 14 non-smoking male chrome-plating workers exposed to Cr(VI) in soluble aerosol form who did not report any significant current or past respiratory disease. Cr-U mainly correlated with Cr-P (Cr in plasma) at the end of the working shift (r(2) = 0.59, p < 0.01), whereas Cr-RBC correlated with EBC-Cr (r(2) = 0.32, p < 0.05); at the beginning of the shift, the only significant correlation was between Cr-U and Cr-RBC (r(2) = 0.74, p < 0.01). The clearance of Cr(iii) arising from Cr(VI) reduction was rapid, thus making Cr-U and Cr-P ideal biomarkers of the most recent exposure, whereas Cr-RBC may represent the fraction of Cr(VI) that reaches the bloodstream in non-reduced form and therefore depends on the airway inhaled dose represented by EBC-Cr. Cr-RBC clearance is slower and not only involves the free diffusion of Cr(iii) from RBC to plasma, but probably also involves more complicated kinetic phenomena involving other tissues and organs, which may explain the correlation between Cr-RBC and Cr-U and the lack of correlation Cr-RBC and Cr-P at least 36 h after the last exposure. In conclusion, our findings reinforce the idea that measuring Cr in EBC can significantly contribute to traditional biomonitoring by providing specific information at the target organ level and integrating our knowledge of Cr toxicokinetics.

Determination of aldehydes in exhaled breath of patients with lung cancer by means of on-fiber-derivatisation SPME-GC/MS.

A number of volatile organic compounds (VOCs) have been identified and used in preliminary clinical studies of the early diagnosis of lung cancer. The aim of this study was to evaluate the potential of aldehydes (known biomarkers of oxidative stress) in the diagnosis of patients with non-small cell lung cancer (NSCLC). We used an on-fiber-derivatisation SPME sampling technique coupled with GC/MS analysis to measure straight aldehydes C3-C9 in exhaled breath. Linearity was established over two orders of magnitude (range: 3.3-333.3×10(-12) M); the LOD and LOQ of all the aldehydes were respectively 1×10(-12) M and 3×10(-12) M. Accuracy was within 93% and precision calculated as % RSD was 7.2-15.1%. Aldehyde stability in a Bio-VOC(®) tube stored at +4°C was 10-17 h, but this became >10 days using a specific fiber storage device. Finally, exhaled aldehydes were measured in 38 asymptomatic non-smokers (controls) and 40 NSCLC patients. The levels of all of the aldehydes were increased in the NSCLC patients without any significant effect of smoking habits and little effect of age. The good discriminant power of the aldehyde pattern (90%) was confirmed by multivariate analysis. These results show that straight aldehydes may be promising biomarkers associated with NSCLC, and increase the sensitivity and specificity of previously identified VOC patterns.

Development and set-up of a portable device to monitor airway exhalation and deposition of particulate matter.

The aim of this study was to assess and monitor airway exhalation and deposition of particulate matter (PM). After standardizing inspiratory/expiratory flow and volumes, a novel device was tested on a group of 20 volunteers and in a field study on workers exposed to cristobalite. Both male and female subjects showed a higher percentage of deposition in the 0.5 microm channel than in the 0.3 microm channel on a laser particle counter, but it was higher in the males because of their higher exhaled lung volumes. The device was tested on a wider range of particles (0.3-0.5-1.0-2.5 microm) in the cristobalite productive division. The device has low intrasubject variability and good reproducibility, with geometric mean of %CV < 5%. Such a measure can be used to assess individual susceptibility to PM, making repeated measures in different environments, and examining the persistence of particles in the airways after a period in polluted environments.

Metallic elements in exhaled breath condensate and serum of patients with exacerbation of chronic obstructive pulmonary disease.

Biomarkers in exacerbated chronic obstructive pulmonary disease may be useful in aiding diagnosis, defining specific phenotypes of disease, monitoring the disease and evaluating the effects of drugs. The aim of this study was the characterization of metallic elements in exhaled breath condensate and serum as novel biomarkers of exposure and susceptibility in exacerbated chronic obstructive pulmonary disease using reference analytical techniques. C-Reactive protein and procalcitonin were assessed as previously validated diagnostic and prognostic biomarkers which have been associated with disease exacerbation, thus useful as a basis of comparison with metal levels. Exhaled breath condensate and serum were obtained in 28 patients at the beginning of an episode of disease exacerbation and when they recovered. Trace elements and toxic metals were measured by inductively coupled plasma-mass spectrometry. Serum biomarkers were measured by immunoassay. Exhaled manganese and magnesium levels were influenced by exacerbation of chronic obstructive pulmonary disease, an increase in their concentrations--respectively by 20 and 50%--being observed at exacerbation in comparison with values obtained at recovery; serum elemental composition was not modified by exacerbation; serum levels of C-reactive protein and procalcitonin at exacerbation were higher than values at recovery. In outpatients who experienced a mild-moderate chronic obstructive pulmonary disease exacerbation, manganese and magnesium levels in exhaled breath condensate are elevated at admission in comparison with values at recovery, whereas no other changes were observed in metallic elements at both the pulmonary and systemic level.

Metallic elements in exhaled breath condensate of patients with interstitial lung diseases.

Epidemiological data support the hypothesis that environmental and occupational agents play an important role in the development of interstitial lung diseases such as idiopathic interstitial pneumonia (IIPs) and sarcoidosis. The aim of this study was to assess the elemental composition of exhaled breath condensate (EBC) in patients with interstitial lung diseases (ILDs) of unknown etiology and healthy subjects as an indirect evaluation of tissue burden, which could improve our understanding of the role of metals in the pathogenesis of ILDs. EBC was obtained from 33 healthy subjects, 22 patients with sarcoidosis, 15 patients with non-specific interstitial pneumonia (NSIP) and 19 with IIPs. Trace elements and toxic metals in the samples were measured by means of inductively coupled plasma-mass spectrometry. There are only small overall differences in the EBC levels of a number of metallic elements among patients with idiopathic pulmonary fibrosis (IPF), NSIP or sarcoidosis, and no pattern is capable of distinguishing them with a high degree of sensitivity and specificity. However, a pattern of pneumotoxic (Si, Ni) and essential elements (Zn, Se and Cu) with the addition of Co distinguished the patients with ILDs from healthy non-smokers with relatively high degrees of sensitivity (96.4%) and specificity (90.9%). Assessing the elemental composition of EBC in patients with different ILDs seems to provide useful information. The non-invasiveness of the EBC method makes it suitable for patients with pulmonary diseases, although further studies are required to confirm the usefulness of this approach and to better understand the underlying pathophysiological processes.

Metallic elements in pulmonary biopsies from lung cancer and control subjects.

Occupational/environmental exposure to some metallic elements is a risk factor for the development of lung diseases, including lung cancer. We aimed at investigating the levels of arsenic, beryllium, cadmium, cobalt, chromium, nickel and lead in the lung tissue of patients affected by early stage non small cell lung cancer (NSCLC). A small number of patients without a diagnosis of lung cancer were also included as control group. Lung tissue biopsies were collected from 45 NSCLC patients (both cancerous and unaffected tissues) and 8 control subjects undergoing surgery. Patients were stratified for smoking habits, histopathology and cancer sites. Metallic elements were determined in dry tissue after digestion by means of ICP-MS. Cd, Ni and Pb levels were higher in unaffected than in control tissues (0.52 vs 0.18 microg/g dry, p < 0.05 for Cd; 4.49 vs 1.8 microg/g dry,p < 0.05 for Ni; 0.21 vs 0.06 microg/g dry, p < 0.01 for Pb). The three elements, and particularly Cd, were influenced by smoking habits; Pb levels were higher in squamocellular carcinoma than adenocarcinomas; Ni distributed in the lungs in an inhomogeneous way. This study demonstrates that the unaffected lung tissue is more representative than the cancerous tissue of the pulmonary content of metallic elements. Tobacco smoke is a main factor affecting the concentration levels of Cd, Pb, and to a lesser extent Ni in the lung tissues of NSCLC patients. The role of past environmental-occupational exposures could not be fully elucidated, due to the limited sample size and the retrospective nature of the study.

Breath analysis in non small cell lung cancer patients after surgical tumour resection.

Exhaled volatile organic compounds (VOCs), mainly aliphatic and aromatic hydrocarbons, have been proposed as a diagnostic test for early lung cancer detection, but the effect of lung cancer surgical re-moval on exhaled VOCs pattern has never been specifically addressed. The aim of this study was to compare VOC levels measured in non small cell lung cancer (NSCLC) patients before surgery (T0), one month (T1) and 3 years (T2) after surgical removal of tumour. In order to better understand the pathophysiological meaning of exhaled aromatic hydrocarbons, the same exhaled biomarkers were also assessed in cancerous and macroscopically unaffected lung tissue samples collected during surgical operation. Exhaled breath was collected in a specially designed Teflon bulb trapping the last 150 ml of a single slow vital capacity. After solid phase micro-extraction, VOCs were analysed in gas chromatography-mass spectrometry. VOC levels were unaffected by surgical removal, except for isoprene, whose concentration was significantly reduced. Three years after surgical operation, some VOCs significantly changed from baseline: in particular, we noted a decrease in isoprene and benzene concentrations, whereas the levels of pentane, toluene and ethylbenzene were increased in comparison with baseline values. Finally, lung tissue analysis showed that all aromatic hydrocarbons, except xylenes, were significantly higher in cancerous than in unaffected tissue. This study showed that surgical operation can influence the concentration of some exhaled VOCs opening a new scenario in the use of exhaled VOCs in lung cancer patients, not only for diagnostic but also for follow up purposes.

Urea in exhaled breath condensate of uraemics and patients with chronic airway diseases.

Exhaled breath condensate (EBC) is composed mainly by water and also contains non-volatile mediators, which are expired in small droplets of airway fluid. Urea has been proposed as a normalization factor for EBC non-volatile biomarkers. Aim of this study was to assess volatility and diffusivity of urea ex vivo and to measure its EBC concentrations in different clinical conditions. Volatility was assessed quantifying EBC concentrations collected at 4 different temperatures, whereas diffusivity was tested by measuring urea concentrations in both plasma and EBC from uraemic patients on intermittent haemodialysis. Urea was also measured in EBC from patients with chronic airway diseases, i.e., chronic obstructive pulmonary disease, asthma, and cystic fibrosis. The concentration of urea but not its absolute amount in EBC increased with condensation temperature. Haemodialysis influenced EBC and plasma urea concentrations in a similar way. The concentrations of urea in chronic airway diseases did not significantly differ from those of controls. Urea is a non-volatile molecule ex vivo and EBC urea depends on its concentrations in plasma. Urea concentrations in EBC are unaffected by three chronic airway diseases. We suggest that there is no need to normalize non-volatile biomarkers in EBC for urea concentrations to account for inter-individual variability. However, in repeated measurements within the same individual, the use of urea either as a normalizing factor or as covariate variable could be proposed to control intra-individual variability.

[Non-invasive assessment of lung pathobiology in hairdressers]. / Valutazione non invasiva della patobiologia polmonare in una coorte di lavoratori del settore acconciatura.

BACKGROUND: Hairdressers are exposed to chemical agents with potentially irritant and sensitizing effects on airways. OBJECTIVES: To asses the presence of respiratory symptoms and biochemical and functional changes of the airways in a cohort of hairdressers. METHODS: Respiratory symptoms, lung function tests, fractional exhaled nitric oxide (FE(NO)50) and selected oxidative stress biomarkers [hydrogen peroxide (H2O2), malondialdehyde (MDA) and 4-hydroxynonenal (HNE)] in exhaled breath condensate (EBC) were assessed in 23 hairdressers on a rest day (Group 1); 12 workers (Group 2) were willing to perform the same tests at the beginning (BS) and at the end of a shift (ES) on the same working day. Eighteen subjects not occupationally exposed to airways irritants formed the control group. RESULTS: Most of the hairdressers reported respiratory symptoms during work; however, all (except one) showed normal spirometry indexes. FE(NO)50 levels were within the reference limits and did not change comparing BS vs. ES sampling. H2O2 and HNE values in EBC were higher in Group 1 (H2O2: 0,16 [0,05-0,19] microM; HNE: 0,94 [0,82-1,22] nM) than in controls (H2O2: 0,05 [0,02-0,09] microM; HNE: 0,61 [0,49-0,78] nM, p < 0,001). In Group 2, H2O2 and MDA levels were higher in EBC collected at ES (0,56 [0,23-3,62] mM and 5,21 [4,93-5,95] nM) in comparison with the BS values (0,11 [0,03-0,28] mM and 4,12 [3,46-5,16] nM, p < 0,001 and p < 0,02, respectively). CONCLUSIONS: Increased levels of oxidative stress biomarkers are detectable in EBC of hairdressers, without impairment in respiratory function. Exhaled biomarkers of oxidative stress may be sensitive end points for evaluating early biochemical changes in the airways of hairdressers.

The effect of inhaled chromium on different exhaled breath condensate biomarkers among chrome-plating workers.

Chromium is corrosive, cytotoxic, and carcinogenic for humans and can induce acute and chronic lung tissue toxicity. The aim of this study was to investigate Cr levels in exhaled breath condensate (EBC) of workers exposed to Cr(VI) and to assess their relationship with biochemical changes in the airways by analyzing EBC biomarkers of oxidative stress, namely, hydrogen peroxide (H2O2) and malondialdehyde (MDA). EBC samples were collected from 24 chrome-plating workers employed in a chrome-plating plant both before and after the Friday work shift and before the work shift on the following Monday. Cr-EBC levels increased from the beginning (5.3 microg/L) to the end of Friday (6.4 microg/L) but were considerably lower on Monday morning (2.8 microg/L). A similar trend was observed for H2O2-EBC levels (which increased from 0.36 microM to 0.59 microM on Friday and were 0.19 microM on Monday morning) and MDA-EBC levels (which increased from 8.2 nM to 9.7 nM on Friday and were 6.6 nM on Monday). Cr-EBC levels correlated with those of H2O2-EBC (r = 0.54, p < 0.01) and MDA-EBC (r = 0.59, p < 0.01), as well as with urinary Cr levels (r = 0.25, p < 0.05). The results of this study demonstrate that EBC is a suitable matrix that can be used to investigate both Cr levels and biomarkers of free radical production sampling the epithelial-lining fluid of workers exposed to Cr(VI).

Exhaled volatile organic compounds in patients with non-small cell lung cancer: cross sectional and nested short-term follow-up study.

BACKGROUND: Non-invasive diagnostic strategies aimed at identifying biomarkers of lung cancer are of great interest for early cancer detection. The aim of this study was to set up a new method for identifying and quantifying volatile organic compounds (VOCs) in exhaled air of patients with non-small cells lung cancer (NSCLC), by comparing the levels with those obtained from healthy smokers and non-smokers, and patients with chronic obstructive pulmonary disease. The VOC collection and analyses were repeated three weeks after the NSCLC patients underwent lung surgery. METHODS: The subjects' breath was collected in a Teflon bulb that traps the last portion of single slow vital capacity. The 13 VOCs selected for this study were concentrated using a solid phase microextraction technique and subsequently analysed by means of gas cromatography/mass spectrometry. RESULTS: The levels of the selected VOCs ranged from 10(-12) M for styrene to 10(-9) M for isoprene. None of VOCs alone discriminated the study groups, and so it was not possible to identify one single chemical compound as a specific lung cancer biomarker. However, multinomial logistic regression analysis showed that VOC profile can correctly classify about 80% of cases. Only isoprene and decane levels significantly decreased after surgery. CONCLUSION: As the combination of the 13 VOCs allowed the correct classification of the cases into groups, together with conventional diagnostic approaches, VOC analysis could be used as a complementary test for the early diagnosis of lung cancer. Its possible use in the follow-up of operated patients cannot be recommended on the basis of the results of our short-term nested study.

Exhaled breath condensate as a suitable matrix to assess lung dose and effects in workers exposed to cobalt and tungsten.

The aim of the present study was to investigate whether exhaled breath condensate (EBC), a fluid formed by cooling exhaled air, can be used as a suitable matrix to assess target tissue dose and effects of inhaled cobalt and tungsten, using EBC malondialdehyde (MDA) as a biomarker of pulmonary oxidative stress. Thirty-three workers exposed to Co and W in workshops producing either diamond tools or hard-metal mechanical parts participated in this study. Two EBC and urinary samples were collected: one before and one at the end of the work shift. Controls were selected among nonexposed workers. Co, W, and MDA in EBC were analyzed with analytical methods based on mass spectrometric reference techniques. In the EBC from controls, Co was detectable at ultratrace levels, whereas W was undetectable. In exposed workers, EBC Co ranged from a few to several hundred nanomoles per liter. Corresponding W levels ranged from undetectable to several tens of nanomoles per liter. A parallel trend was observed for much higher urinary levels. Both Co and W in biological media were higher at the end of the work shift in comparison with preexposure values. In EBC, MDA levels were increased depending on Co concentration and were enhanced by coexposure to W. Such a correlation between EBC MDA and both Co and W levels was not observed with urinary concentration of either element. These results suggest the potential usefulness of EBC to complete and integrate biomonitoring and health surveillance procedures among workers exposed to mixtures of transition elements and hard metals. Key words: cobalt, exhaled breath condensate, hard metals, lung, malondialdehyde, oxidative stress, tungsten.