Sample Collection

Bronchial brushings of normal-appearing airway collected cross-sectionally from high- risk subjects with and without PMLs profiled by RNA-Seq

Autofluorescence bronchoscopy was performed to obtain bronchial airway brushings from subjects enrolled in the British Columbia Lung Health Study (BC-LHS) at the British Columbia Cancer Agency (BCCA) (Vancouver, BC) between June 2000 and March 2011130. In addition, during the procedure PMLs were sampled (if present) and evaluated by a team of pathologists. Histological lesion grade was assigned to each sampled PML and the worst histology observed was recorded and assigned to the corresponding, normal-appearing brushing. The study participants with normal or hyperplasia histology enrolled in the BC-LHS were current or recent former smokers between 50 and 75 years old with no prior history of lung cancer, who smoked for at least 20 years and whose estimated 3-year lung cancer risk was at least 2%. Baseline bronchial brushes were collected from subjects with evidence of PMLs enrolled in multiple

chemoprevention studies, were current or recent former smokers between 40 and 79 years old with no prior history of lung cancer and at least 30 pack years (i.e. having smoked 1 pack a day for 30 years).

Bronchial brushes of normal-appearing epithelium from 84 BCCA subjects (1 brush from each subject) with and without PMLs were selected to undergo mRNA-Seq

while ensuring balanced clinical covariates, such as age, pack years, race, sex, and COPD status.

The data is available from NCBI’s Gene Expression Omnibus (GEO) using the accession ID GSE79315.

Bronchial brushings of normal-appearing airway collected longitudinally from high-risk subjects with history of PMLs profiled by RNA-Seq

Additional bronchial airway brushings were obtained from subjects participating in the High-Risk Lung Cancer-Screening Program at Roswell Park Cancer Institute (RPCI) (Buffalo, NY) between December 2009 and March 2013. These subjects were at high risk for developing lung cancer by either having a prior history of lung or

aerodigestive cancers or by being a current or recent former smoker at least 50 years of age, with at least 20 smoked pack years. Fifty-one bronchial brushes of normal-appearing epithelium from 23 RPCI subjects with and without PMLs were profiled by mRNA-Seq (18 subjects had 2 procedures and 5 subjects had 3 procedures). Samples were classified as stable/progressive if the worst histological grade at the second time point for a given patient remained the same or worsened, and regressive if the worst histological grade at the second time point improved. The RPCI samples were utilized in biomarker validation to evaluate its power to identify subjects with progressing lesions by calculating

differences in the biomarker score between sequential procedures. The data is available as part of GSE79315.

Bronchial brushings of normal-appearing airway collected cross-sectionally from subjects with and without COPD and PMLs profiled by microarrays

A total of 238 bronchial airway brishings from current and former smokers with and without COPD and PMLs were profiled on Affymetrix Human Gene 1.0 ST Array as described in Steiling et al 122 and used as an external source of validation samples to further evaluate the biomarker’s ability to detect PMLs. CEL files with mRNA expression were downloaded from GEO (GSE37147) and processed by Dr. Jennifer Beane using Robust Multi-array Average (RMA) 53 and the Ensembl Gene CDF v16.0.0 file

(http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/16.0.0/ensg.asp

) to standardize gene annotation. Fourty four samples were filtered out based on sex mismatches and quality. A subset of 36 samples profiled on microarrays as part of the study originated from BCCA subjects and was used as an overlapping validation set. The remaining 158 bronchial airway brushings obtained and profiled in the same manner from additional subjects at high risk of developing lung cancer, were used as an independent validation set.

Bronchial brushings of normal-appearing airway collected cross-sectionally from subjects with and without lung cancer and profiled by microarrays.

Current and former smokers with suspect lung cancer underwent flexible bronchoscopy as part of two additional microarray studies. A total of 164 samples

in GEO as GSE4115. A total of 299 samples described by Silvestri et al 114 _{were profiled} on Affymetrix Human Gene 1.0 ST Array and deposited in GEO as GSE66499. These extra bronchial brushing datasets were downloaded from GEO in CEL format and processed using Robust Multi-array Average (RMA) 53 and the Ensembl Gene CDF v16.0.0 file

(http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/16.0.0/ensg.asp

) to standardize gene annotation. Both studies were used to validate the biomarker’s ability to distinguish brushings from subjects with and without lung cancer.

Tumor and adjacent normal biopsies collected cross-sectionally from subjects with lung squamous cell carcinoma and profiled by RNA-Seq.

Tumor (n=502) and matched adjacent normal (n=51) samples collected from subjects with squamous cell lung cancer were profiled by mRNA-Seq by The Cancer Genome Atlas (TCGA) Research Network Team 137. RSEM-normalized log2-transformed counts along with the corresponding clinical data were downloaded from the UCSC Xenabrowser, which provides access to TCGA’s Genomic Data Commons (GDC) Data Portal, and used to evaluate the biomarker’s ability to distinguish normal from tumor samples originating from subjects diagnosed with lung cancer.

The Institutional Review Boards (IRBs) of all participating institutions approved the studies and all subjects provided written informed consent.