Early Detection of Lung Cancer by Molecular Markers in Endobronchial Epithelial Lining Fluid Nicolas Kahn, Michael Meister, Ralf Eberhardt, Thomas Muley, Philipp A. Schnabel, Christian Bender, Marc Johannes, Denise Keitel, Holger Sültmann, Felix JF. Herth, Ruprecht Kuner Online Data Supplement 1. Methods 2. Supplementary Table 1 - 5 Methods Patient Cohort This study included 71 patients with indeterminate solitary pulmonary nodules (IPN) who were scheduled to undergo diagnostic bronchoscopy at the Thoraxklinik Heidelberg between 2007 and 2010. The Institutional Review Board approved the data collection and analysis, and written informed consent was obtained from all patients prior to the procedure. Definitive histological diagnosis was established either by transbronchial biopsy or by subsequent surgical resection, and included adenocarcinoma (AC, n=31), squamous cell carcinoma (SCC, n=14), large cell lung carcinoma (LCLC, n=4), mixed type (n=1), carcinoid (n=1), benign nodule (n=20). Patient characteristics and ELF sampling parameters are given in Table 1. Wilcoxon, Fisher’s exact and Chi-square tests were used to compare parameters associated with the diagnosis of nodules. Patients were separated into a screening (n=15) and a validation cohort (n=56) (Figure 1). Detailed description of each patient and the experimental assignment is listed in Table E1. Bronchoscopic Microsampling In summary, the procedure was performed under general anesthesia or conscious sedation in a standardized fashion. A standard BAL catheter was placed through the working channel in peripheral lung and sampling of endobronchial lining fluid was performed by using a specific mucus collection probe (Olympus, BC-401C/BC402C). The bronchoscopic microsampling device (BMS) was inserted through the guide catheter into the bronchi, positioned fluoroscopically, and placed at the nearest possible point next to the lesion. The collection probe containing the absorbed ELF was sectioned and immediately shock-frozen in liquid nitrogen. In each patient, BMS procedures were repeated twice at the site of the lesion (IPN) and at the corresponding subsegmental bronchus of the contralateral lung (CL) as internal control. RNA extraction and microarray experiment Total RNA extraction of ELF samples was performed using RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. The quantity of total RNA was measured by spectrophotometer and the quality was assessed by capillary electrophoresis (Agilent Technologies GmbH, Waldbronn, Germany). For microarray experiments, we selected total RNA samples from nodules and contralateral site, which yielded higher concentration and quality (total amount of RNA > 200ng, RIN value > 5). We used the microarray platform Affymetrix GeneChip U133 Plus 2.0 (Affymetrix, Santa Clara, CA, US) covering about 47,000 transcripts and variants. The microarray experiment comprised 30 ELF samples of matched nodule and contralateral site (15 individuals). Total RNA (150 ng) was amplified using GeneChip® 3’ IVT Express Kit (Affymetrix). Labeling, hybridization and scanning was processed according to the manufacturers’ instructions (Affymetrix). The microarray data were normalized by the gcRMA method as previously described (1), and deposited into the NCBI GEO database (GSE27489). Linear models for microarray data software package (LIMMA) (2) was used to identify differentially expressed genes between ELF samples of malignant (or AC and SCC respectively) and benign nodules dependent (ratio IPN/ CL) or independent (IPN only) of the contralateral site. The genes were ranked according to the LIMMA score and by the linear fold change. Quantitative real-time PCR In the validation study, qRT-PCR was used to analyze gene expression in 112 independent specimens including matched indeterminate pulmonary nodule and contralateral ELF extracts of the same patients (n = 56). Total RNA was reversely transcribed including oligo d(T) and random primers (50 pmol each) with the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Burlington, ON, Canada). Expression analysis of 16 genes was done for each sample in technical duplicates by using the quantitative real-time PCR system (ABI Prism 7900HT Sequence Detection System; Applied Biosystems, Weiterstadt, Germany) and gene specific primers and probes (Taqman assays, ABI). Relative quantification was done using two internal control genes esterase D (ESD) and polymerases 2A (POLR2A) as described (1). A detailed list of examined genes is given in Table E2. The qRT-PCR data were deposited into the NCBI GEO database (GSE27553). Statistical analysis was done using the paired or unpaired Wilcoxon test, respectively. Genes were regarded to be differentially expressed with a p-value < 0.05 and a fold change < 0.5 or > 2. We accounted for the interaction between biomarker expression and significant clinical parameters (nodule diameter, age) by using ANOVA analysis. The discriminative efficacy of various parameters (nodule diameter, TNC expression and multiplying both values) was analysed by receiver operating characteristics (ROC) curves using the area under the curve (AUC) as a measure. All p-values were derived from 2-tailed statistical tests, and a value of 0.05 or less was regarded as statistically significant. The microarray and the qRT-PCR data were deposited MIAME-compliant into the NCBI GEO database as super series (GSE27554). References E1 Kuner R, Muley T, Meister M, Ruschhaupt M, Buness A, Xu EC, Schnabel P, Warth A, Poustka A, Sültmann H, Hoffmann H. Global gene expression analysis reveals specific patterns of cell junctions in non-small cell lung cancer subtypes. Lung Cancer 2009;63:32-38. E2 Smyth GK. Linear models and empirical Bayes methods for assessment differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3, 2004;No. 1, Article 3. Table E1: Demographics for 71 patients including diagnosis, TNM status, nodule diameter and location, sampling distance to the nodule, and the assignment to the experimental series (microarray screening, qRT-PCR validation). Pt. Smoking Diameter Tumor Sampling Distance Age Gender Diagnosis TNM No (Pack Years) in mm Location to Nodule 9 72 M 40 Adeno T2N2M0 57 LLL < 20mm 18 64 M 45 Adeno T1N1M1 40 RML < 20mm 22 67 F 0 Adeno T2N0M0 59 RLL < 20mm 32 61 M 40 Adeno T2N2M0 43 RUL < 20mm 33 51 F 35 Adeno T1N0M1 20 LUL > 20mm 34 74 M 0 SCC T2N0M0 74 LLL < 20mm 36 72 F 30 SCC T2N0M0 22 RLL < 20mm 38 68 M 60 SCC T1N0M0 24 RUL < 20mm 41 75 M n/a SCC T2N0M0 38 RLL < 20mm 56 63 M 80 SCC T2N1M0 30 RUL < 20mm 23 37 F 15 benign n/a 35 RML < 20mm 47 64 M 35 benign n/a n/a n/a n/a 51 61 F 0 benign n/a 25 LLL < 20mm 53 79 F 0 benign n/a 22 LLL > 20mm 59 49 M 15 benign n/a 23 RUL < 20mm 6 54 F 60 Adeno T4N1M0 13 RUL > 20mm 7 73 M n/a Adeno T1N1M0 28 RUL < 20mm 8 51 F 20 Adeno T2N0M1 43 RUL < 20mm 12 59 F 30 Adeno T1N0M0 28 RML < 20mm 15 75 M 60 Adeno T1N0M0 30 RUL < 20mm 16 75 F 15 Adeno T2N0M0 45 RUL < 20mm 19 74 M 100 Adeno T1N0M0 25 LUL > 20mm 21 69 M 6 Adeno T2N0M0 47 LUL < 20mm 25 57 M 60 Adeno T2N2M1 43 LLL < 20mm 27 60 M 60 Adeno T1N0M0 19 RLL < 20mm 28 70 F 0 Adeno T1N1M0 28 LLL < 20mm 31 67 M 0 Adeno T2N0M0 58 RML > 20mm 35 70 F 10 Adeno T1N0M0 25 LUL < 20mm 39 68 F n/a Adeno T2N2M1 16 LLL < 20mm 52 64 F 0 Adeno T1N0M0 27 LUL > 20mm 54 77 M 20 Adeno T2N0M0 41 RUL < 20mm 60 73 F 0 Adeno T2N0M0 25 RUL > 20mm 61 60 F n/a Adeno T2N2M1 32 LUL > 20mm 64 58 M 100 Adeno T4N0M0 37 LLL > 20mm 65 68 F 45 Adeno T1N0M0 16 LLL < 20mm 69 67 F 70 Adeno T2N1M0 42 LUL < 20mm 70 63 M 50 Adeno T2N0M0 22 RLL < 20mm 73 69 M 20 Adeno T2N1M0 33 LLL < 20mm 75 71 M 40 Adeno T1N0M0 32 RML < 20mm 76 74 F 0 Adeno T1N2M0 30 LLL < 20mm 77 68 F 0 Adeno T2N0M0 38 LLL < 20mm 1 69 M 35 SCC T4N0M0 24 RLL < 20mm 4 65 M 80 SCC T2N2M0 40 RLL < 20mm 5 75 M 40 SCC T2N0M0 57 RUL < 20mm 10 72 M 40 SCC T2N0M0 18 LUL < 20mm 24 66 M 30 SCC T2N1M0 49 RLL < 20mm 29 67 M 100 SCC T1N2M1 35 LLL < 20mm 30 62 M 30 SCC T2N3M1 19 RUL > 20mm 48 74 F 40 SCC T2N0M1 19 RUL > 20mm 71 70 M 50 SCC T2N0M0 55 LLL < 20mm 3 68 M 30 LCLC T1N2M1 27 LUL < 20mm 17 50 F 5 Carcinoid T1N0M0 30 RUL < 20mm 20 69 M 90 LCLC T1N0M0 33 RUL < 20mm 42 68 M n/a LCLC T2N2M0 28 RUL < 20mm 43 72 F 70 LCLC T2N0M0 42 LLL < 20mm 72 45 F 50 NSCLC T1N0M0 41 RUL < 20mm 2 57 F 10 benign n/a 14 LUL < 20mm 11 73 F n/a benign n/a 22 RLL < 20mm 13 72 F 0 benign n/a 21 RML < 20mm 14 75 M 45 benign n/a 16 RUL < 20mm 26 63 M 80 benign n/a 14 LUL < 20mm 37 71 M 40 benign n/a 18 RLL > 20mm 40 58 F 40 benign n/a 16 RLL > 20mm 50 65 M n/a benign n/a 23 RUL < 20mm 55 50 F 35 benign n/a 25 LLL > 20mm 57 47 M 25 benign n/a 21 LUL < 20mm 62 55 F 0 benign n/a 35 RLL > 20mm 66 48 F 30 benign n/a 23 RLL < 20mm 67 50 M 34 benign n/a 26 RML < 20mm 68 64 F 45 benign n/a 23 RLL < 20mm Experiment Microarray Screening Microarray Screening Microarray Screening Microarray Screening Microarray Screening Microarray Screening Microarray Screening Microarray Screening Microarray Screening Microarray Screening Microarray Screening Microarray Screening Microarray Screening Microarray Screening Microarray Screening qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation qRT-PCR Validation Table E2: Summary of qPCR-measured genes including nomenclature, expression range, absent values (Ct > 40) and standard deviation range across technical duplicates. Gene Symbol Gene ID AssayID B2M CXCL14 ESD IFITM1 KRT17 MMP9 MYC POLR2A S100A8 S100A9 SDC1 SERPINB13 SFN SULF2 TNC TP63 All Genes 567 9547 2098 8519 3872 4318 4609 5430 6279 6280 6382 5275 2810 55959 3371 8626 Hs99999907_m1 Hs00171135_m1 Hs00382661_m1 Hs01652522_g1 Hs01588578_m1 Hs00234579_m1 Hs00153408_m1 Hs00172187_m1 Hs00374263_m1 Hs00610058_m1 Hs00896423_m1 Hs00202640_m1 Hs00968567_s1 Hs00378697_m1 Hs01115665_m1 Hs00978340_m1 Median Ct value Median Ct value Ct IPN - Ct CL Absent Values STABW > 0.5 IPN CL 0 / 112 22.6 21.2 1.5 0 / 112 0 / 112 31.1 30.1 1.1 6 / 112 0 / 112 1.6 2 / 112 28.6 27.0 0 / 112 28.3 28.0 0.3 2 / 112 1 / 112 29.6 28.3 1.2 2 / 112 1 / 112 -1.0 44 / 112 33.9 34.9 0 / 112 30.5 29.7 0.8 5 / 112 0 / 112 1.0 1 / 112 29.2 28.1 0 / 112 28.4 29.5 -1.1 1 / 112 0 / 112 28.2 29.1 -0.8 5 / 112 2 / 112 30.0 29.1 0.9 4 / 112 1 / 112 33.1 31.5 1.6 21 / 112 0 / 112 30.0 29.2 0.8 4 / 112 2 / 112 31.2 29.9 1.3 7 / 112 1 / 112 30.8 30.0 0.8 5 / 112 1 / 112 29.3 28.0 1.3 3 / 112 9 / 1792 Median Ct 29.8 Median Ct 29.1 1.0 112 / 1792 Table E3: List of the 120 genes best ranked in six different LIMMA analyses. The results included group-wise comparison (NSCLC subtypes adenocarcinoma and squamous cell carcinoma, and all together, versus benign samples) only on the nodule site (IPN) and both the nodule and contralateral site (IPN/CL). SYMBOL CXCL14 AZGP1 TNC CDH2 IER3 XDH APOOL GJB2 SDC1 SDC1 MYC ADAMDEC1 NA MARCKSL1 COCH PGGT1B NA NA TPD52 CSNK1G3 SERPINB13 SERPINB5 KRT5 TNC SERPINB13 DSC3 GABRP KRT17 GPR87 VTCN1 FHL2 UGT1A1 FGFBP1 KRT17 PHLDA2 PROM2 TFCP2L1 UGT1A6 CLCA4 UGT1A9 TNC SERPINB5 GABRP UGT1A@ FHL2 SERPINB13 PHLDA2 DSC3 PROM2 UGT1A6 FAM110C SDC1 SFN SDC1 IER3 SFN LAPTM4B MYC NOL3 LOC389023 ID logFC 222484_s_at 3.55004394 209309_at 2.95948957 201645_at 2.61307197 203440_at 1.62292496 201631_s_at 1.51087882 241994_at 1.21004906 244187_at 1.18566348 223278_at 1.17372708 201287_s_at 1.10854616 201286_at 1.10748242 202431_s_at 1.0326155 206134_at 1.00206396 230958_s_at 0.89519567 200644_at 0.88778952 1554241_at 0.85062697 242844_at 0.84236585 235531_at 0.80754394 235438_at 0.74769244 201691_s_at 0.74717868 220768_s_at 0.73089693 217272_s_at -3.15445171 204855_at -3.04274 201820_at -2.97476908 201645_at -2.60730735 211361_s_at -2.56642111 206032_at -2.56343652 205044_at -2.54546311 205157_s_at -2.47936066 219936_s_at -2.39228716 219768_at -2.30359195 202949_s_at -2.2889413 207126_x_at -2.22150866 205014_at -2.20484491 212236_x_at -2.0936005 209803_s_at -2.06344674 1552797_s_at-1.99585771 227642_at -1.98683686 206094_x_at -1.93460024 220026_at -1.87839761 204532_x_at -1.84611342 201645_at 2.61018966 204855_at 2.2975384 205044_at 2.29219268 215125_s_at 2.23064841 202949_s_at 1.98930569 211361_s_at 1.96795452 209803_s_at 1.85926379 206032_at 1.81876561 1552797_s_at1.74671067 206094_x_at 1.5875259 226863_at 1.38341131 201287_s_at 1.38007217 33323_r_at 1.3667934 201286_at 1.33985384 201631_s_at 1.33784761 33322_i_at 1.31122961 208029_s_at 1.2614713 202431_s_at 1.21384107 221566_s_at 1.12332203 236351_at 1.08124322 Fold change 11.7130423 7.77848706 6.11805032 3.07998848 2.84983584 2.31345504 2.2746798 2.25593747 2.15628243 2.15469314 2.04572964 2.0028633 1.85986212 1.85033888 1.80328443 1.79298803 1.75022929 1.67910499 1.67850714 1.6596706 8.9039884 8.2405464 7.86130639 6.09365302 5.92338192 5.91114053 5.83795509 5.57650285 5.24988986 4.9368539 4.88697356 4.66380883 4.61024974 4.2681193 4.17983717 3.98853161 3.96367001 3.82272188 3.6766647 3.59530314 6.10583949 4.91618226 4.89799968 4.69344876 3.97045871 3.91213057 3.62822465 3.52779228 3.35592545 3.00533517 2.60884514 2.60281392 2.57896715 2.53125673 2.52773918 2.48152951 2.39740111 2.31954377 2.17848025 2.1158586 P.Value 0.003996108 0.003087153 0.000834666 0.007952892 0.003199845 0.002102334 0.002629694 0.007115853 0.002396455 0.002441288 0.006267785 0.00797927 0.007190832 0.008093465 0.008102736 0.00787552 0.004282167 0.006208031 0.004751792 0.005743842 0.000372364 0.000118473 0.003217369 0.000849275 0.000258785 4.52E-05 0.002492441 0.002455201 0.000703292 0.001812874 0.004719307 0.004867298 0.000191124 0.001621023 0.003149021 0.0023313 0.00395545 0.003040648 0.00361236 0.005098701 0.000142254 0.001517662 0.00144676 0.004359395 0.00396027 0.001854619 0.001836776 0.002695294 0.001841993 0.004679693 0.003518665 0.000212032 0.002395231 0.000214096 0.002232067 0.00236228 0.002688079 0.00066 0.002506077 0.001983519 Comparison IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN Adeno vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN SCC vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign IPN Tumor vs Benign continued Table E3: List of the 120 genes best ranked in six different LIMMA analyses. The results included group-wise comparison (NSCLC subtypes adenocarcinoma and squamous cell carcinoma, and all together, versus benign samples) only on the nodule site (IPN) and both the nodule and contralateral site (IPN/CL). CXCL14 CXCL14 MMP10 IGFBP3 IFITM1 IGFBP3 TP63 S100A2 IFITM1 TNC KRT15 KRT5 PHLDA2 CFH PROM2 FMO3 SULF2 SDC1 MARCKSL1 ARL4C KRT5 SFN SPINK5 PTPRZ1 LOC642587 KRT17 SULF2 SFN UGT1A6 TFCP2L1 SDC1 LAYN PLEKHH1 IL20RB SEMA3F RHBDF1 SH3RF2 MCF2L LTB4R SLC1A5 SERPINB13 TP63 S100A2 KRT5 KRT15 IGFBP3 DST KRT17 SULF2 SFN KRT17 PROM2 FGFR3 PTPRZ1 SDC1 SFN ARL4C MT1X LOC642587 SDC1 222484_s_at 4.41341281 218002_s_at 3.87777882 205680_at 3.20392514 212143_s_at 2.84766726 214022_s_at 2.74702434 210095_s_at 2.60665221 209863_s_at 2.59258196 204268_at 2.50075962 201601_x_at 2.40803191 201645_at 2.40724484 204734_at 2.13614616 201820_at 2.08423933 209803_s_at 2.07943972 213800_at 2.02769889 1552797_s_at1.80181067 40665_at 1.78612336 224724_at 1.71628331 201286_at 1.66215602 200644_at 1.6444801 202207_at 1.61900178 201820_at -2.26042421 33323_r_at -1.7168327 205185_at -1.6404787 204469_at -1.63828328 226755_at -1.62782236 212236_x_at -1.62747123 224724_at -1.56811959 33322_i_at -1.50060374 206094_x_at -1.48696814 227642_at -1.44503205 201286_at -1.37108621 228080_at -1.35991329 225726_s_at -1.336768 228575_at -1.18582495 35666_at -1.07427589 218686_s_at -0.93809394 243582_at -0.91362948 35147_at -0.90470729 236172_at -0.74503965 208916_at -0.72757779 217272_s_at 2.3066891 209863_s_at 2.29468906 204268_at 2.2709979 201820_at 2.17233177 204734_at 2.07814204 210095_s_at 1.97040582 204455_at 1.94898564 205157_s_at 1.68589976 224724_at 1.64220145 33323_r_at 1.64197662 212236_x_at 1.60299749 1552797_s_at1.59528016 204379_s_at 1.57045134 204469_at 1.52923063 201286_at 1.51662112 33322_i_at 1.49223628 202207_at 1.42217628 204326_x_at 1.35439805 226755_at 1.33873982 201287_s_at 1.33124955 21.3093223 14.7003522 9.21462296 7.19835503 6.71331037 6.09088646 6.03177229 5.65983354 5.30749796 5.3046032 4.39586221 4.24051454 4.22643048 4.0775396 3.48657537 3.44886907 3.285888 3.16489146 3.12635174 3.07162433 4.79132346 3.28713954 3.11769262 3.11295188 3.09046165 3.08970956 2.9651798 2.82961101 2.802993 2.72268871 2.58665243 2.56669752 2.52584831 2.27493441 2.10566494 1.9159952 1.88377868 1.87216461 1.67602033 1.65585666 4.94746362 4.90648232 4.82656865 4.50751337 4.22263059 3.91878335 3.86102966 3.21740994 3.12141774 3.12093134 3.0377381 3.02153187 2.96997613 2.88631875 2.86120153 2.81324712 2.67989463 2.5569041 2.52930289 2.51620514 0.000952602 0.003846747 0.004524985 0.002103822 0.002200323 0.000635091 0.000788029 0.001987341 0.000664743 0.000970476 0.003414637 0.00341274 0.003561383 0.004368545 0.002556344 0.004498405 0.000279894 0.00021009 0.001026156 0.003444755 0.001932157 0.00097619 0.001004443 0.004319302 0.002509732 0.001332843 0.000596368 0.001454568 0.003025771 0.004649587 0.00103346 0.003306171 0.004711424 0.002670182 0.001804976 0.001686339 0.002049217 0.003787732 0.004602117 0.003206719 0.003459181 0.000553495 0.001042945 0.000556283 0.000964248 0.003866336 0.001876961 0.002853613 6.50E-05 0.000267608 0.000283188 0.001783559 0.001954879 0.001842054 9.43E-05 0.00028713 0.002613635 0.001262922 0.003887763 0.000658355 IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL Adeno vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL SCC vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign IPN/CL Tumor vs Benign Table E4: Differentially expressed genes in ELF samples between nodules and the contralateral site of each patient with malignant and non-malignant diagnosis (IPN vs. CL). Gene S100A8 $ MMP9 $ S100A9 $ IFITM1 $ TNC $ CXCL14 $ SULF2 MYC SERPINB13 SDC1 KRT17 TP63 SFN B2M IPN vs CL Tumor paired p-value† ratio 0.003294 3.18 0.004710 3.76 0.011070 1.49 0.001388 1.70 0.010200 1.69 0.038130 1.74 0.142300 1.23 0.051400 1.14 0.194200 0.72 0.917600 1.20 0.798500 0.97 0.189400 0.84 0.984000 1.00 0.537900 1.05 IPN vs CL Adeno paired p-value† ratio 0.059350 3.25 0.015780 3.31 0.101400 2.26 0.001972 2.03 0.056430 1.54 0.045120 1.39 0.140900 1.21 0.352500 1.03 0.064650 0.68 0.691500 1.27 0.877400 1.05 0.218200 0.94 0.366600 0.89 0.129100 1.17 Legend: † Wilcoxon test, $ significant genes (p-value < 0.05; fold change > 2) IPN vs CL SCC paired p-value† ratio 0.027340 2.69 0.562500 6.40 0.039060 2.73 0.359400 0.99 0.019530 4.68 0.039060 8.26 0.312500 1.39 0.054690 2.94 0.910200 1.66 0.546900 2.17 0.425800 1.80 0.910200 1.59 0.054690 2.26 0.164100 0.60 IPN vs CL benign paired p-value† ratio 0.001526 9.14 0.053710 3.60 0.010740 2.89 0.025570 1.25 0.168800 0.72 0.241200 0.71 0.488700 0.96 0.421200 1.00 0.357500 0.93 0.389400 0.75 0.207800 0.85 0.151400 0.97 0.421200 0.88 0.025570 1.35 Table E5: Differentially expressed genes in ELF samples of the contralateral site (CL) with malignant and non-malignant diagnosis. Gene S100A9 $ KRT17 IFITM1 SFN SERPINB13 SDC1 SULF2 S100A8 CXCL14 TNC TP63 MYC MMP9 B2M CL Tumor vs Benign p-value† ratio 0.075530 2.64 0.264000 1.43 0.441200 1.07 0.633400 1.00 0.459500 1.51 0.878600 0.89 0.455500 1.07 0.769600 1.34 0.633400 1.04 0.633400 0.97 0.962700 0.93 0.918100 0.91 0.830800 1.23 0.063850 1.48 CL Adeno vs Benign p-value† ratio 0.221000 1.64 0.561800 1.22 0.698100 1.04 0.406500 1.04 0.173100 1.57 0.803800 0.84 0.390900 1.08 0.738100 1.42 0.719700 1.12 0.580600 0.97 0.890300 0.96 0.658600 0.99 0.665400 1.40 0.028120 1.54 Legend: † Wilcoxon test, $ significant genes (p-value < 0.05; fold change > 2) CL SCC vs Benign p-value† ratio 0.040860 4.05 0.215200 1.31 0.173700 1.42 0.289500 0.69 0.411500 0.84 0.825200 0.79 1.000000 0.95 0.860900 1.34 0.482000 0.81 0.558400 0.44 0.770200 0.75 0.238400 0.52 0.462300 1.33 0.215200 1.57 CL Adeno vs SCC p-value† ratio 0.445900 0.40 0.489100 0.93 0.423900 0.73 0.040820 1.50 0.021570 1.87 0.983600 1.07 0.789000 1.14 0.515700 1.06 0.591000 1.38 0.140000 2.19 0.564700 1.28 0.065890 1.91 0.235500 1.05 0.752700 0.98