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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - EXACT (Identifying biomarkers of Exposure leading to Lung Cancer with Adductomics)

Teaser

Summary

Lung Cancer (LC) is the leading cause of cancer death worldwide (1.8 million deaths in 2018), the survival of patients is highly depended on accurate and early diagnosis, and current methods are reported to partially result in diagnostic error resulting in treatment delay and decrease of survival rates. Further knowledge is needed to detect early markers of lung cancer and improve understanding of early carcinogenesis. Lung Cancer is known to be causally linked to tobacco smoke and recently air pollution has also been recognized as a lung carcinogen. To understand the mechanisms of LC onset, methods are needed to characterize specific toxicants in tobacco smoke and air pollution that people are exposed to, as well as molecular pathways. Electrophiles present in blood are highly reactive species that have been long suspected of causing cancer and other diseases. These electrophiles enter the organism by direct exposure (inhalation, ingestion) or via internal mechanisms such as metabolism of exogenous compounds, oxidation of lipids, and inflammation associated with prior disease and contribute to oxidative stress. Oxidative stress (OS) has been linked to smoking habits and is known to contribute to carcinogenesis. Reactive oxygen species (ROS) contribute to oxidative stress, causing cell damage, but as they are small and rapidly degraded, they cannot be easily quantified. These highly reactive species have been long suspected of causing cancer and other diseases because of their ability to bind to DNA and proteins. The challenge for their measurement is due to their very short half-life in the body and their high reactivity, which makes them almost impossible to measure. However, it is possible to measure the adducts resulting from their binding to DNA or proteins (Hemoglobin and Albumin). The Cysteine34 (Cys34) locus of the Human Serum Albumin (HSA) represents a preferred reaction site for small electrophilic species. HSA has a long half-life in serum (30 days) and is present at high concentration; therefore, it is a good marker to measure exposure to toxic electrophiles. Up to now, current techniques have focussed on the targeted measure of pre-selected albumin-adducts in serum to detect exposure to certain chemicals. Although targeted assays can relate Cys34 adducts to specific exposures, they are not well suited for discovering unknown causes of human diseases arising from exposures to electrophilic species. In this project, we applied a new method based on high resolution mass spectrometry; this novel approach is based on an agnostic untargeted analysis of known and unknown adducts of albumin and electrophiles to determine oxidative stress.
The objectives of EXACT were to (i) Apply agnostic adductomics to a nested case-control study of 250 prospective cases and controls of Lung Cancer (ii) Determine differential adducts profiles in cases and controls years before diagnosis (iii) Relate adduct profiles to exposure data and methylation measurements in order to elucidate biological pathways and exposure leading to lung cancer.

Conclusions: For the first time we were able detect, in prospective blood samples, decreased levels of the N-acetylcysteine (NAC) and Cysteine-Glycine adducts in lung cancer cases. Altogether, our results highlight the implications of these adducts in the oxidative stress response contributing to lung cancer years before diagnosis.

Work performed

We measured human serum albumin (HSA) untargeted adductomics profiles in 250 plasma samples from the prospective lung cancer cases (median follow up 7 years) and healthy controls from the Italian component of the European Prospective Investigation into Cancer and Nutrition (EPIC-Italy) cohort. To determine the implication of tobacco smoke, we used detailed information on smoking status, smoking intensity, time since cessation and the comprehensive smoking index. The levels of exposure to air pollution at participantsâ€™ homes were estimated via land-use regression models. Individual levels for 2 gases: NO2 and NOx, fine particles with a diameter of 2.5 Âµg/m3 (PM2.5), and 10 Âµg/m3 (PM10) were estimated. We evaluated the association between each given adduct and (i) exposure to tobacco smoke, (ii) exposure to air pollution and (iii) prospective lung cancer status using linear mixed models. Our models accounted for several important characteristics of the participants such as gender, age, centre of recruitment and BMI as well as technical variables. In order to further understand the role of exposure to tobacco smoke in lung carcinogenesis with adductomics, we explored the association linking adducts levels to 2670 assayed methylation CpG sites that were determined as epigenetics markers smoking in previous work.

We identified 42 (including 14 novel) HSA adducts. Our analysis of lung cancer outcome identified 6 differentially abundant adducts in prospective cases compared to controls (p<0.05). Among these, the adduct of N-acetylcysteine, a powerful anti-oxidant, showed the stronger association, with lower levels in future cases than controls. The lower abundance of the NAC adduct in cases may reflect the lower bioavailability of NAC in serum suggestive of dysregulation of redox control. As we disposed of a very limited number of non-smoking Lung Cancer cases, only very week associations of adducts levels and exposure to air pollution were observed. The level of the NAC adduct was associated to the methylation levels of 11 smoking-related CpG sites. Our findings are consistent with a perturbation in redox pathways and consequently a potential imbalance in ROS levels and elimination in future lung cancer cases.
Altogether this approach provides interesting new insights in lung carcinogenesis and highlights the ability of HSA adductomics to explore the biological processes by which oxidative stress and redox balance dysregulation may contribute, years before clinical manifestations, to carcinogenesis and to investigate the role of smoking in these processes

The developments and findings of EXACT have been published in 1 peer-reviewed journal article, 1 edited book, 2 international conferences and 2 invited talks. Additional publications are currently planned (2 submitted, 1 in preparation). The results of EXACT have been presented to the REA in the context of the EXPOSOMICS project. EXACT has contributed to public outreach activities, the project has been presented at the Imperial College Festival 2017 and 2018, the MRC Festival 2017 and Imperial Fringe 2017 reaching out to the general public and school students.

Final results

This project showed the interest of the use of a unique novel adductomics approach coupled with methylation analysis for the understanding of mechanisms related to oxidative stress in future Lung Cancer cases years before diagnosis. This technique has been applied for the first time in these types of samples. The development and validation of a biomarker combining a stable measure of oxidative stress with methylation of DNA can allow the identification of subjects particularly susceptible to lung carcinogenesis. The biomarker could be used to monitor the success of anti-smoking activities, of air pollution reduction campaigns and of dietary supplementation and thus help preventing lung cancer in high-risk individuals, thus reducing healthcare costs.