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