**2. Materials and methods**

The people that were interested and met the criteria for the inclusion factors were enrolled as voluntaries (Figure 1).

**Figure 1.** Diagram of the enrolling process for recruiting the voluntaries for LC prevention.

The high risk subjects for LC were recruited via advertisement in newspaper, radio and internet (University of Chile web) and were included in this study from two regions of Chile, Metropolitan (Santiago city) and Antofagasta. Enrolment was based on a LC Risk Survey (Witteman et al., 2011) by Washington University School of Medicine, which was modified by our group. Subjects were eligible for enrolment procedure if they met the following criteria's: male/female aged 40 years or older; family history of LC, non-smokers and ever smokers exposed naturally to environmental air pollution (Metropolitan region) or to arsenic in drinking water (Antofagasta region) for at least 10 years.

invasive lesions in the voluntaries with high likehood of malignance for LC, according to AQC

The people that were interested and met the criteria for the inclusion factors were enrolled as

**Figure 1.** Diagram of the enrolling process for recruiting the voluntaries for LC prevention.

The high risk subjects for LC were recruited via advertisement in newspaper, radio and internet (University of Chile web) and were included in this study from two regions of Chile, Metropolitan (Santiago city) and Antofagasta. Enrolment was based on a LC Risk Survey (Witteman et al., 2011) by Washington University School of Medicine, which was modified by our group. Subjects were eligible for enrolment procedure if they met the following criteria's: male/female aged 40 years or older; family history of LC, non-smokers and ever smokers

and DR70.

**2. Materials and methods**

64 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

voluntaries (Figure 1).

In addition, we enrolled subjects who were suspected of having lung cancer (N=12) based on their clinical symptoms, with the final diagnosis completed in the study. These patients did not have previous tests performed on them such as cytology or CT and did not have any treatment. However, only 12 of them were in agreement to undergo to the AFB, while all of them underwent a CT to rule out or confirm the LC.

The voluntaries were classified according to their risk score, which allowed for classification of the subjects in three risk categories: low, medium and high. Subjects with medium or high risk were invited to participate in the study. The LC risk predictor was applied to 508 volunteer participants, obtaining 364 subjects with medium to high-risk for LC. Of those, 224 were from Santiago and 140 were from the Antofagasta's region.

Sputum generation was induced by inhalation of 3% saline solution, after administration of two puffs of salbutamol. Patients were instructed to cough and expectorate before their Specimens were collected in 50 mL centrifuge tubes, followed by adding enough SedFix solution. Each tube was mixed with DTT solution and was shaken at 1800 rpm overnight. After centrifugation, a cell pellet was obtained; re suspended in an ethanol solution and was placed on slides. The cell DNA was stained with Feulgen thionin and was analysed by an automated cytometry-based scoring system, as described by Kemp et al. (Kemp et al, 2007).

Furthermore, blood samples were taken in serum separator tubes in the morning before ingestion of any meal. The tubes were left at room temperature for 30 minutes. The serum was separated from the cells by centrifugation at 3,500 rpm for 10 min. Each serum specimen was diluted 1:200 with the diluent buffer and was tested along with the calibrators according to AMDL Diagnostics Onko Sure protocol (Radient Pharmaceuticals) as described in Adonis et al. (2005) and Hatton et al. (2006).

Participants with positive DR70 test (threshold>1.0), or an AQC score ≥4.6 were invited to have an AFB, using the Onco-LIFE device (Novadaq Inc., Richmond, Canada), under local anaes‐ thesia. The airways were examined by, White Light Broncoscopy (WLB) and Auto Fluores‐ cence Broncoscopy (AFB) and the visual findings were classified as normal, abnormal or suspicious, as described by Lam et al. (1998). Endobronchial mucosal biopsies were taken from all areas that were suspicious under WLB or AFB. In addition, surveillance biopsies were taken from epithelium with normal appearance in all subjects. An average of 2-3 biopsies was taken from each of the participants.

Categorical variables were analysed by Fisher's exact test. Sensitivity, specificity, likeli‐ hood ratio (LR), Predictive Positive Value (PPV) and Predictive Negative Value (PNV) were calculated with 95% CI. Statistical significance was accepted at p<0.05. The sensitivity and specificity were assessed for each test and was used in analysis as a single test; two-test parallel combination and two-test series combination (Sullivan and Thomson, 2000, Kruskal, 1998).
