Demographic characteristics according to endoscopic BE and pathological BE in the training cohort^*.

<p>^*Data shown are mean (SD) for continuous variables and number (percentage) for categorical variables</p>†<p>P for trend test.</p

ROC curve for a) endoscopically visible CLE of any length independent of histology (AUC: 0.61), b) segment containing IM≥2 cm (AUC: 0.64) in the external validation cohort (N = 477).

<p>ROCs curve were developed using the risk scores which are calculated by the weights of different predictors. The weights were developed based on the coefficients of predictors in the backward logistic regression model in the training cohort.</p

ROC curve for a) endoscopically visible CLE of any length independent of histology (AUC: 0.72), b) segment containing IM≥2 cm (AUC: 0.81) in the training cohort (N = 1603).

<p>ROCs curve were developed using the risk scores which are calculated using the weights of different predictors. The weights were developed based on the coefficients of predictors in the backward logistic regression model in the training cohort.</p

The coefficients, weights and odds ratios of selected predictors for BE.

<p>^*n/a: not applicable.</p

Summary of the predictors for BE in the external validation cohort^*.

<p>^*Data shown are mean (SD) for continuous variables and number (percentage) for categorical variables.</p

TLR pathway mutations in EAC.

<p>The EAC samples with TLR pathway mutations in the (A) ICGC cohort (n = 171 patients) and (B) TCGA cohort (n = 149 patients), with gene names along the vertical axis and sample names along the horizontal axis. The mutation status for known driver mutations in EAC (<i>TP53</i>, <i>CDKN2A</i>, <i>SMAD4</i>, <i>PIK3CA</i>) is presented for each sample with TLR pathway mutations in the ICGC and TCGA cohorts. Recurring oncogenic amplifications (<i>ERBB2</i>, <i>EGFR</i>, <i>CCND1</i>) and deletions (<i>CDKN2A/B</i>) that are known drivers in EAC are presented for the ICGC cohort. LY96 is the gene encoding MD2 protein. (C) Total TLR pathway mutation frequency from the combined ICGC and TCGA cohorts (n = 320), with gene names along the vertical axis and percent of mutated samples along the horizontal axis. (D) Schematic of the TLR signaling pathway summarizing the somatic mutations from the combined ICGC and TCGA cohorts, with mutated pathway components highlighted in color and large bold font. Grey pathway components have no TLR pathway mutations in either cohort.</p

Distribution of TLR pathway mutations in EAC and other cancers.

<p>(A) TLR pathway mutations in different cancer types from the COSMIC database, with cancer types along the vertical axis and percent of mutated samples along the horizontal axis (abbreviations provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006808#pgen.1006808.s003" target="_blank">S3 Table</a>). <i>TLR4</i> mutations are indicated in dark blue. (B) Schematic of TLR4 protein with mutations from the ICGC (green circles) and TCGA (purple circles) EAC cohorts. Both EAC cohorts identified missense mutations at amino acid position E439 and F487. (C) Schematic of TLR4 protein with mutations from different cancer types in the COSMIC database. (D) Three lanes show dimer contacts (to other TLR4 chain), MD2, and LPS (PDB ID: 4G8A [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006808#pgen.1006808.ref022" target="_blank">22</a>]). The fourth lane was calculated from a homology model of TLR4 TIR domain dimer (based on PDB ID: 2J67 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006808#pgen.1006808.ref023" target="_blank">23</a>]). Contacting TLR4 residues with atoms less than 0.5 Å apart at surfaces were determined using Chimera findclash function. Numbers of contacts are plotted in 10 residue sections of increasingly dark red tones (maximum 8 is black).</p

Clinicopathologic characteristics grouped by TLR pathway mutation status.

<p>Clinicopathologic characteristics grouped by TLR pathway mutation status.</p

Functional analysis of <i>TLR4</i> mutations.

<p>(A) NF-κB response to TLR4 ligands in HEK293 cells transfected with <i>TLR4</i> mutants <i>versus</i> wild-type <i>TLR4</i> and stimulated with 100 ng/ml synthetic Lipid A and 10ng/ml LPS. The y-axis is normalized luminescence, calculated by dividing NF-κB firefly luciferase by the housekeeper renilla luciferase. Results are the average of three experiments with all conditions performed in triplicate. Error bars are standard deviation. *p<0.05, **p<0.01, ***p<0.001. (B) TLR4 protein expression 48 hours after transfection for NF-κB luciferase reporter assays. Representative Western blot shows protein expression for nine <i>TLR4</i> mutants, the double mutant (E439G + F703C), wild-type <i>TLR4</i> and empty vector. Fully glycosylated TLR4 is the upper band (120 kDa), and deglycosylated TLR4 is the lower band (110 kDa). (C) Immunofluorescence staining FLAG-AlexaFluor 488 antibody (green) in HEK293 cells transfected with wild-type TLR4-FLAG shows cytoplasmic distribution, in comparison to empty vector. Mutant E439G and R787H did not show any difference in localization of TLR4 protein. DAPI (blue) and Phalloidin (red) were used to visualize the cell nucleus and cytoskeleton, respectively. (D) Fold-change secretion of IL-8 for the EAC cell lines OE33 and JH-EsoAd1 following transfection of <i>TLR4</i> mutants E439G, R787H, E439G+F703C and wild type <i>TLR4</i>. Cells were stimulated with synthetic MPLA and LPS for 24 hours prior to ELISA. Data were normalized by dividing the concentration of IL-8 by the unstimulated (nil) value for each transfection condition. Data are an average of four independent experiments performed in duplicate. Error bars are standard deviation. *p<0.05, **p<0.01,</p