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Supplementary Tables Table S1. Early efffcts of NNK exposure on the mouse airway transcriptome. Table S2. Evolutionarily conserved decreased gene expression in the airway following NNK exposure. Table S3. Genes differentially regulated in the mouse airway at the indicated timepoints post NNK exposure. Table S4. Mouse genes downregulated as early as T2 vs T0 and enriched in airways of human smokers with lung cancer relative to cancer-free smokers. Table S5. Pathways analysis of Mouse genes enriched in airways of human smokers with lung cancer and modulated during in vivo lung oncogenesis

Supplementary Figure S1 Details. Expression levels of 384 genes selected for the NMF algorithm in the three KRAS-mutant LUAC subsets.

Supplementary Methods and Supplementary Figure Legends

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand–receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment.Significance:The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception.This article is highlighted in the In This Issue feature, p. 2355

PDF file - 4670K, Sweave report depicting complete codes use for microarray analysis and identification of genes differentially expressed in the molecular field of injury after excluding main carinas from the analysis

PDF file - 129K, Sweave report depicting complete codes use for microarray analysis of paired adjacent and contralateral airways

Supplementary Methods

Perspective on this Article from Knockout of the Tumor Suppressor Gene Gprc5a in Mice Leads to NF-κB Activation in Airway Epithelium and Promotes Lung Inflammation and Tumorigenesis

Supplementary Table S1. Individual KRAS, STK11/LKB1, TP53, ATM and KEAP1 somatic mutations from the indicated clinical cohorts.

S1. Lung tumor development in NNK-exposed Gprc5a-/- mice. S2. Evolutionarily conserved patterns of tobacco-carcinogen mediated gene expression in murine and human airway epithelia. S3. Early and persistent changes in mouse airway gene expression in vivo following tobacco carcinogen exposure and during lung oncogenesis. S4. Gene profiles downregulated in the mouse normal-appearing airway field of injury are overall suppressed in lung tumors relative to adjacent normal lung.

Lung cancer continues to be a major deadly malignancy. The mortality of this disease could be reduced by improving the ability to predict cancer patients' survival. We hypothesized that genes differentially expressed among cells constituting an in vitro human lung carcinogenesis model consisting of normal, immortalized, transformed, and tumorigenic bronchial epithelial cells are relevant to the clinical outcome of non–small cell lung cancer (NSCLC). Multidimensional scaling, microarray, and functional pathways analyses of the transcriptomes of the above cells were done and combined with integrative genomics to incorporate the microarray data with published NSCLC data sets. Up-regulated (n = 301) and down-regulated genes (n = 358) displayed expression level variation across the in vitro model with progressive changes in cancer-related molecular functions. A subset of these genes (n = 584) separated lung adenocarcinoma clinical samples (n = 361) into two clusters with significant survival differences. Six genes, UBE2C, TPX2, MCM2, MCM6, FEN1, and SFN, selected by functional array analysis, were also effective in prognosis. The mRNA and protein levels of one these genes—UBE2C—were significantly up-regulated in NSCLC tissue relative to normal lung and increased progressively in lung lesions. Moreover, stage I NSCLC patients with positive UBE2C expression exhibited significantly poorer overall and progression-free survival than patients with negative expression. Our studies with this in vitro model have lead to the identification of a robust six-gene signature, which may be valuable for predicting the survival of lung adenocarcinoma patients. Moreover, one of those genes, UBE2C, seems to be a powerful biomarker for NSCLC survival prediction.

Effects of waterpipe smoking on lung pathobiology and carcinogenesis remain sparse despite the worldwide emergence of this tobacco vector. To address this gap, we investigated the effects of chronic waterpipe smoke (WPS) exposure on lung pathobiology, host immunity, and tumorigenesis using an experimental animal model that is prone to tobacco carcinogens and an exploratory observational analysis of human waterpipe smokers and nonsmokers. Mice exhibited elevated incidence of lung tumors following heavy WPS exposure (5 days/week for 20 weeks) compared to littermates with light WPS (once/week for 20 weeks) or control air. Lungs of mice exposed to heavy WPS showed augmented CD8+ and CD4+ T cell counts along with elevated protumor immune phenotypes including increased IL17A in T/B cells, PD-L1 on tumor and immune cells, and the proinflammatory cytokine IL1β in myeloid cells. RNA-sequencing (RNA-seq) analysis showed reduced antitumor immune gene signatures in animals exposed to heavy WPS relative to control air. We also performed RNA-seq analysis of airway epithelia from bronchial brushings of cancer-free waterpipe smokers and nonsmokers undergoing diagnostic bronchoscopy. Transcriptomes of normal airway cells in waterpipe smokers, relative to waterpipe nonsmokers, harbored gene programs that were associated with poor clinical outcomes in patients with lung adenocarcinoma, alluding to a WPS-associated molecular injury, like that established in response to cigarette smoking. Our findings support the notion that WPS exhibits carcinogenic effects and constitutes a possible risk factor for lung cancer as well as warrant future studies that can guide evidence-based policies for mitigating waterpipe smoking.Prevention Relevance:Potential carcinogenic effects of waterpipe smoking are very poorly understood despite its emergence as a socially acceptable form of smoking. Our work highlights carcinogenic effects of waterpipe smoking in the lung and, thus, accentuate the need for inclusion of individuals with exclusive waterpipe smoking in prevention and smoking cessation studies.

XLSX file - 68K, Supplementary table depicting 1165 genes significantly differentially expressed by site in the molecular field of injury

XLSX file - 65K, Supplementary table exhibiting genes significantly differentially expressed by site in the molecular field of injury when excluding main carinas from the analysis

Supplementary Data from Distinct Immune Gene Programs Associated with Host Tumor Immunity, Neoadjuvant Chemotherapy, and Chemoimmunotherapy in Resectable NSCLC

PDF file 87K, Summary of multivariate analysis of outcome in lung adenocarcinoma patients by combined expression of EZH2 and TTF-1

Supplementary Information - PDF file 109K, Legends for Supplementary Figures 1-11

PDF file - 53K, Antibodies used for immunohistochemistry.

Table S1: Samples analyzed by DNA and RNA sequencing; Table S2: DNA (n=67) and RNA (n=48) sequencing quality metrics for each sample; Table S3: Exonic mutations in known lung adenocarcinoma drivers and other cancer associated genes; Table S4: Genes mutated in atypical adenomatous hyperplasias and lung adenocarcinomas; Table S5: Validation of specific BRAF, KRAS and EGFR mutations using digital PCR; Table S6: Genes differentially expressed among normal lung tissues, atypical adenomatous hyperplasias and lung adenocarcinomas; Table S7: Genes differentially expressed between BRAF-mutant, KRAS-mutant and BRAF/KRAS wild type atypical adenomatous hyperplasias; Table S8: Markers of immune signaling that are aberrantly expressed between normal lung tissues, atypical adenomatous hyperplasias and lung adenocarcinomas.

Microphotographs of representative examples of the computer algorithm (Aperio GENIE) used to analyze the IHC PD-L1 expression (brown staining) in malignant cells (MCs) and tumor associated macrophages (TAMs). (A) Whole histology section image of a representative ADC stained with PD-L1 IHC processed using the Aperio GENIE image analysis algorithm depicting areas tumor, stroma and macrophages infiltration. B) Higher power images of H&E (left upper), CD68 (right upper), PD-L1 positive expression in tumor MCs (left lower), and illustration of the GENIE algorithm for membrane staining and the computer generated data on intensity and extension of IHC expression (right lower). C) Higher power images of H&E (left upper), CD68 (right upper), PD-L1 positive expression in tumor TAMs (left lower) and illustration of the GENIE algorithm for membrane staining and the computer generated data on intensity and extension of IHC expression (right lower).

Purpose: Enhancer of zeste homolog 2 (EZH2) promotes carcinogenesis by epigenetically silencing tumor suppressor genes. We studied EZH2 expression by immunohistochemistry in a large series of non–small cell lung carcinomas (NSCLC) in association with tumor characteristics and patient outcomes.Experimental Design: EZH2 immunohistochemistry expression was analyzed in 265 normal and premalignant bronchial epithelia, 541 primary NSCLCs [221 squamous cell carcinomas (SCC) and 320 adenocarcinomas] and 36 NSCLCs with paired brain metastases. An independent set of 91 adenocarcinomas was also examined. EZH2 expression was statistically correlated with clinico-pathological information, and EGFR/KRAS mutation status.Results: EZH2 expression was significantly (P < 0.0001) higher in SCCs compared with adenocarcinomas and in brain metastasis relative to matched primary tumors (P = 0.0013). EZH2 expression was significantly (P < 0.0001) elevated in bronchial preneoplastic lesions with increasing severity. In adenocarcinomas, higher EZH2 expression significantly correlated with younger age, cigarette smoking, and higher TNM stage (P = 0.02 to P < 0.0001). Higher EZH2 expression in adenocarcinoma was associated with worse recurrence-free survival (RFS; P = 0.025; HR = 1.54) and overall survival (OS; P = 0.0002; HR = 1.96). Furthermore, lung adenocarcinomas with low EZH2 levels and high expression of the lineage-specific transcription factor, TTF-1, exhibited significantly improved RFS (P = 0.009; HR = 0.51) and OS (P = 0.0011; HR = 0.45), which was confirmed in the independent set of 91 adenocarcinomas.Conclusion: In lung, EZH2 expression is involved in early pathogenesis of SCC and correlates with a more aggressive tumor behavior of adenocarcinoma. When EZH2 and TTF-1 expressions are considered together, they serve as a prognostic marker in patients with surgically resected lung adenocarcinomas. Clin Cancer Res; 19(23); 6556–65. ©2013 AACR.

PDF file - 63K, Calibration curves for RFS and OS.

Supplementary Figure from Distinct Immune Gene Programs Associated with Host Tumor Immunity, Neoadjuvant Chemotherapy, and Chemoimmunotherapy in Resectable NSCLC

Supplementary Table 1 - PDF file 57K, Clinicopathological information of the NSCLC tissue microarray specimens analyzed by IHC analysis

Supplementary Tables 1-3 Supplementary Table 1. Location and number of NSCLC, normal and field of cancerization specimens analyzed for genome-wide allelic imbalance Supplementary Table 2. Summary of identified allelic imbalance events in the normal-appearing airway field of cancerization in early-stage NSCLC Supplementary Table 3. Airway events with negative B-allele frequency correlations

Supplementary Figures S1-S2; Supplementary Tables S1-S5.

Purpose: To identify the pattern of interleukin-1 receptor–associated kinase (IRAK-1) protein expression in non–small cell lung carcinoma (NSCLC) and corresponding preneoplastic lesions.Experimental Design: Archived tissue from NSCLC (adenocarcinoma and squamous cell carcinoma; n = 306) and adjacent bronchial epithelial specimens (n = 315) were analyzed for the immunohistochemical expression of IRAK-1, and the findings were correlated with patients' clinicopathologic features. Furthermore, we investigated the correlation between IRAK-1 expression and expression of NF-κB and IL-1α in tumor specimens.Results: NSCLC tumors showed significantly higher cytoplasmic and lower nuclear IRAK-1 expression than normal epithelium. Squamous dysplasias had significantly higher cytoplasmic IRAK-1 expression than normal epithelium. In tumors, a significant positive correlation was detected between IRAK-1 expression (nuclear and cytoplasmic; P = 0.011) and IL-1α cytoplasmic expression (P < 0.0001). The correlation between the expression of the markers and patients' clinicopathologic features varied according to tumor histologic type and sex. High IRAK-1 cytoplasmic expression correlated with worse recurrence-free survival in women with NSCLC [hazard ratio (HR), 2.204; P = 0.033], but not in men. In adenocarcinoma, combined low level of expression of nuclear IRAK-1 and NF-κB correlated significantly with worse overall (HR, 2.485; P = 0.007) and recurrence-free (HR, 3.058; P = 0.006) survivals in stage I/II patients.Conclusions: IRAK-1 is frequently expressed in NSCLC tissue specimens, and this expression is an early phenomenon in the sequential development of lung cancer. IRAK-1 is a novel inflammation-related marker and a potential target for lung cancer chemopreventive strategies. Clin Cancer Res; 16(1); 34–44

Purpose: The ETS2 transcription factor is an evolutionarily conserved gene that is deregulated in cancer. We analyzed the transcriptome of lung adenocarcinomas and normal lung tissue by expression profiling and found that ETS2 was significantly downregulated in adenocarcinomas. In this study, we probed the yet unknown functional role of ETS2 in lung cancer pathogenesis.Experimental Design: Lung adenocarcinomas (n = 80) and normal lung tissues (n = 30) were profiled using the Affymetrix Human Gene 1.0 ST platform. Immunohistochemical (IHC) analysis was conducted to determine ETS2 protein expression in non–small cell lung cancer (NSCLC) histologic tissue specimens (n = 201). Patient clinical outcome, based on ETS2 IHC expression, was statistically assessed using the log-rank and Kaplan–Meier tests. RNA interference and overexpression strategies were used to assess the effects of ETS2 expression on the transcriptome and on various malignant phenotypes.Results:ETS2 expression was significantly reduced in lung adenocarcinomas compared with normal lung (P < 0.001). Low ETS2 IHC expression was a significant predictor of shorter time to recurrence in NSCLC (P = 0.009, HR = 1.89) and adenocarcinoma (P = 0.03, HR = 1.86). Moreover, ETS2 was found to significantly inhibit lung cancer cell growth, migration, and invasion (P < 0.05), and microarray and pathways analysis revealed significant (P < 0.001) activation of the HGF pathway following ETS2 knockdown. In addition, ETS2 was found to suppress MET phosphorylation and knockdown of MET expression significantly attenuated (P < 0.05) cell invasion mediated by ETS2-specific siRNA. Furthermore, knockdown of ETS2 augmented HGF-induced MET phosphorylation, cell migration, and invasion.Conclusion(s): Our findings point to a tumor suppressor role for ETS2 in human NSCLC pathogenesis through inhibition of the MET proto-oncogene. Clin Cancer Res; 19(13); 3383–95. ©2013 AACR.

Purpose: Resistance to VEGFR inhibitors is a major obstacle in the treatment of non–small cell lung cancer (NSCLC). We investigated the cellular mechanisms mediating resistance of NSCLCs to VEGFR tyrosine kinase inhibitors.Experimental Design: We generated murine models of human NSCLC and performed targeted inhibition studies with the VEGFR TKIs cediranib and vandetanib. We used species-specific hybridization of microarrays to compare cancer (human) and stromal (mouse) cell transcriptomes of TKI-sensitive and -resistant tumors. We measured tumor microvascular density and vessel tortuosity to characterize the effects of therapy on the tumor vascular bed. Circulating cytokine and angiogenic factor levels in patients enrolled in VEGFR TKI trials were correlated with clinical outcomes.Results: Murine xenograft models of human lung adenocarcinoma were initially sensitive to VEGFR TKIs, but developed resistance to treatment. Species-specific microarray analysis identified increased expression of stromal-derived hepatocyte growth factor (HGF) as a candidate mediator of TKI resistance and its receptor, c-MET, was activated in cancer cells and tumor-associated stroma. A transient increase in hypoxia-regulated molecules in the initial response phase was followed by adaptive changes resulting in a more tortuous vasculature. Forced HGF expression in cancer cells reduced tumor sensitivity to VEGFR TKIs and produced tumors with tortuous blood vessels. Dual VEGFR/c-MET signaling inhibition delayed the onset of the resistant phenotype and prevented the vascular morphology alterations. In patients with cancer receiving VEGFR TKIs, high pretreatment HGF plasma levels correlated with poorer survival.Conclusions: HGF/c-MET pathway mediates VEGFR inhibitor resistance and vascular remodeling in NSCLC. Clin Cancer Res; 23(18); 5489–501. ©2017 AACR.

PDF file - 84K, Kaplan Meier curves for RFS and OS by marker group for patients with stage I NSCLC.

Supplementary Table 2 - XLSX file 92K, Gene features significantly differentially expressed in lung cancer cells transfected with ETS2-specific siRNA compared to cells transfected with control siRNA

Supplementary Data from Expression of Interleukin-1 Receptor–Associated Kinase-1 in Non–Small Cell Lung Carcinoma and Preneoplastic Lesions

Supplementary information including supplementary methods, supplementary tables and figures as well as supplementary figure legends. Supplementary Figure 1. HapLOH results for matched normal large airway and NSCLC tumor from case 18 Supplementary Figure 2. Distribution of posterior probabilities for airway events called by hapLOH Supplementary Figure 3: Allelic imbalance events detected in normal-appearing airway brushings Supplementary Figure 4. Event classification using B-allele frequencies and log R ratios Supplementary Figure 5. Effect of allelic imbalance on B-allele frequencies Supplementary Figure 6: Distribution of focal and arm airway events

There is a dearth of knowledge about the pathogenesis of premalignant lung lesions, especially for atypical adenomatous hyperplasia (AAH), the only known precursor for the major lung cancer subtype adenocarcinoma (LUAD). In this study, we performed deep DNA and RNA sequencing analyses of a set of AAH, LUAD, and normal tissues. Somatic BRAF variants were found in AAHs from 5 of 22 (23%) patients, 4 of 5 of whom had matched LUAD with driver EGFR mutations. KRAS mutations were present in AAHs from 4 of 22 (18%) of patients. KRAS mutations in AAH were only found in ever-smokers and were exclusive to BRAF-mutant cases. Integrative analysis revealed profiles expressed in KRAS-mutant cases (UBE2C, REL) and BRAF-mutant cases (MAX) of AAH, or common to both sets of cases (suppressed AXL). Gene sets associated with suppressed antitumor (Th1; IL12A, GZMB) and elevated protumor (CCR2, CTLA-4) immune signaling were enriched in AAH development and progression. Our results reveal potentially divergent BRAF or KRAS pathways in AAH as well as immune dysregulation in the pathogenesis of this premalignant lung lesion. Cancer Res; 77(22); 6119–30. ©2017 AACR.

Purpose: Patients with resected non–small cell lung cancer (NSCLC) are at risk for recurrence of disease, but we do not have tools to predict which patients are at highest risk. We set out to create a risk model incorporating both clinical data and biomarkers.Experimental Design: We assembled a comprehensive database with archival tissues and clinical follow-up from patients with NSCLC resected between 2002 and 2005. Twenty-one proteins identified from our preclinical studies as related to lung carcinogenesis were investigated, including pathways related to metabolism, DNA repair, inflammation, and growth factors. Expression of proteins was quantified using immunohistochemistry. Immunohistochemistry was chosen because it is widely available and can be performed on formalin-fixed paraffin-embedded specimens. Cox models were fitted to estimate effects of clinical factors and biomarkers on recurrence-free survival (RFS) and overall survival (OS).Results: A total of 370 patients are included in our analysis. With median follow-up of 5.3 years, median OS is 6.4 years. A total of 209 cases with recurrence or death were observed. Multicovariate risk models for RFS and OS were developed including relevant biomarkers, age, and stage. Increased expression of phospho-adenosine monophosphate-activated protein kinase (pAMPK), phospho-mTOR (pmTOR), epithelial cell adhesion molecule (EpCAM), and calcium/calmodulin-dependent serine protein kinase were significant (P < 0.05) predictors for favorable RFS; insulin receptor, chemokine (C-X-C motif) receptor 2 (CXCR2), and insulin-like growth factor-1 receptor predicted for unfavorable RFS. Significant (P < 0.05) predictors for favorable OS include pAMPK, pmTOR, and EpCAM; CXCR2 and flap structure–specific endonuclease-1 predicted unfavorable OS.Conclusion: We have developed a comprehensive risk model predictive for recurrence in our large retrospective database, which is one of the largest reported series of resected NSCLC. Clin Cancer Res; 20(7); 1946–54. ©2013 AACR.

PDF file - 68K, Multicovariate Cox model for RFS in stage I NSCLC.

Purpose:Our understanding of the immunopathology of resectable non–small cell lung cancer (NSCLC) is still limited. Here, we explore immune programs that inform of tumor immunity and response to neoadjuvant chemotherapy and chemoimmunotherapy in localized NSCLC.Experimental Design:Targeted immune gene sequencing using the HTG Precision Immuno-Oncology panel was performed in localized NSCLCs from three cohorts based on treatment: naïve (n = 190), neoadjuvant chemotherapy (n = 38), and neoadjuvant chemoimmunotherapy (n = 21). Tumor immune microenvironment (TIME) phenotypes were based on the location of CD8+ T cells (inflamed, cold, excluded), tumoral PD-L1 expression (Results:PD-L1–positive tumors exhibited increased signature scores for various lymphoid and myeloid cell subsets (P < 0.05). TIME phenotypes exhibited disparate frequencies by stage, PD-L1 expression, and mutational burden. Inflamed and PD-L1+/TILs+ NSCLCs displayed overall significantly heightened levels of immune signatures, with the excluded group representing an intermediate state. A cytotoxic T-cell signature was associated with favorable survival in neoadjuvant chemotherapy–treated NSCLCs (P < 0.05). Pathologic response to chemoimmunotherapy was positively associated with higher expression of genes involved in immune activation, chemotaxis, as well as T and natural killer cells (P < 0.05 for all). Among the three cohorts, chemoimmunotherapy-treated NSCLCs exhibited the highest scores for various immune cell subsets including T effector and B cells (P < 0.05).Conclusions:Our findings highlight immune gene programs that may underlie host tumor immunity and response to neoadjuvant chemotherapy and chemoimmunotherapy in resectable NSCLC.

Purpose: Emerging evidence suggests that aberrant expression of oncogenes contributes to development of lung malignancy. The thyroid transcription factor 1 (TITF-1) gene functions as a lineage survival gene abnormally expressed in a significant fraction of non–small cell lung cancers (NSCLC), in particular lung adenocarcinomas.Experimental Design: To better characterize TITF-1 abnormality patterns in NSCLC, we studied TITF-1's gene copy number using FISH and quantitative PCR, as well as its protein expression by immunohistochemistry analysis in a tissue microarray comprising surgically resected NSCLC (N = 321) including 204 adenocarcinomas and 117 squamous cell carcinomas (SCC). TITF-1 copy number and protein expression were correlated with patients' clinicopathologic characteristics, and in a subset of adenocarcinomas with EGFR and KRAS mutation status.Results: We found that increased TITF-1 protein expression was prevalent in lung adenocarcinomas only and was significantly associated with female gender (P < 0.001), never-smokers (P = 0.004), presence of EGFR mutations (P = 0.05), and better overall survival (all stages, P = 0.0478; stages I and II, P = 0.002). TITF-1 copy number gain(CNG) was detected by FISH analysis in both adenocarcinomas (18.9%; high CNG, 8.3%) and SCCs (20.1%; high CNG, 3.0%), and correlated significantly with the protein product (P = 0.004) and presence of KRAS mutations (P = 0.008) in lung adenocarcinomas. Moreover, multivariate analysis revealed that TITF-1 copy number gain was an independent predictor of poor survival of NSCLC (P = 0.039).Conclusions: Our integrative study demonstrates that the protein versus genomic patterns of TITF-1 have opposing roles in lung cancer prognosis and may occur preferentially in different subsets of NSCLC patients with distinct oncogene mutations. Clin Cancer Res; 17(8); 2434–43. ©2011 AACR.

Figure S1: Mutation burden in atypical adenomatous hyperplasia and lung adenocarcinoma; Figure S2: Mutation burden in atypical adenomatous hyperplasia and lung adenocarcinoma based on tobacco history; Figure S3: Mutations in cancer driver genes in atypical adenomatous hyperplasia and lung adenocarcinoma; Figure S4: Representative validation of somatic mutations by digital PCR.

Correlation between median density by mm2 of TAICs expressing immune markers and clinicopathological features in lung ADC specimens.

Supplementary Figures 1-11 - PDF file 905K, Supplementary Figure 1. Decreased ETS2 expression in smoker lung adenocarcinomas compared to adenocarcinomas from never-smoker patients. Supplementary Figure 2. ETS2 expression in never-smoker and smoker lung cancer cell lines. Supplementary Figure 3. IHC analysis of ETS2 protein in NSCLC FFPE histological tissue specimens. Supplementary Figure 4. Over-expression of ETS2 decreases lung cancer cell anchorage-dependent and -independent growth. Supplementary Figure 5. Modulation of HGF-mediated gene-interaction network following knockdown of ETS2 in H441 lung cancer cells. Supplementary Figure 6. Increased phospho-MET levels following ETS2 knockdown. Supplementary Figure 7. Over-expression of ETS2 decreases MET phosphorylation in lung cancer cells. Supplementary Figure 8. HGF treatment increases ETS2 levels in lung cancer cells. Supplementary Figure 9. ETS2 inhibits HGF-induced lung cancer cell proliferation and migration. Supplementary Figure 10. IHC analysis of phosphorylated MET protein in NSCLC FFPE histological tissue specimens. Supplementary Figure 11. NSCLC patients with both low ETS2 and high membrane phospho-MET IHC expression exhibit significantly reduced time to recurrence

Microphotographs of representative examples of the algorithms used to analyze TAICs density. A) Representative positive CD3 staining (brown) in an ADC showing five random square areas (1 mm2 each; green outlined boxes) in both intratumoral (right) and peritumoral (left) compartments divided by a red line. B) High power images of CD3 IHC expression (left) and image analysis quantification using the nuclear algorithm (right). C) High power images of CD4 IHC expression (left) and image analysis quantification using the nuclear algorithm (right); the algorithm excluded a macrophage cell from analysis as indicated by the arrows. D) higher power images of granzyme B IHC expression (left) and image analysis quantification using the nuclear algorithm (right); E) High power images of FOXP3 IHC expression (left) and image analysis quantification using the nuclear algorithm (right). F) High power images of CD68 (macrophages) IHC expression (left) and image analysis quantification using the cytoplasmic algorithm (right).

PDF file - 45K, Consort Diagram.

Visually normal cells adjacent to, and extending from, tumors of the lung may carry molecular alterations characteristics of the tumor itself, an effect referred to as airway field of cancerization. This airway field has been postulated as a model for early events in lung cancer pathogenesis. Yet the genomic landscape of somatically acquired molecular alterations in airway epithelia of lung cancer patients has remained unknown. To begin to fill this void, we sought to comprehensively characterize the genomic architecture of chromosomal alterations inducing allelic imbalance (AI) in the airway field of the most common type of lung tumors, non–small cell lung cancer (NSCLC). To do so, we conducted a genome-wide survey of multiple spatially distributed normal-appearing airways, multiregion tumor specimens, and uninvolved normal tissues or blood from 45 patients with early-stage NSCLC. We detected alterations in airway epithelia from 22 patients, with an increased frequency in NSCLCs of squamous histology. Our data also indicated a spatial gradient of AI in samples at closer proximity to the NSCLC. Chromosome 9 displayed the highest levels of AI and comprised recurrent independent events. Furthermore, the airway field AI included oncogenic gains and tumor suppressor losses in known NSCLC drivers. Our results demonstrate that genome-wide AI is common in the airway field of cancerization, providing insights into early events in the pathogenesis of NSCLC that may comprise targets for early treatment and chemoprevention. Cancer Res; 76(13); 3676–83. ©2016 AACR.

PDF file 68K, C-index of Cox proportional hazard models of outcome of lung adenocarcinoma patients

supplemental legend

Supplemental Figures and Tables

Median TAIC density by mm2 in the intratumoral and peritumoral compartments of lung ADC and SqCC specimens.

Purpose: Identification of effective markers for outcome is expected to improve the clinical management of non–small cell lung cancer (NSCLC). Here, we assessed in NSCLC the prognostic efficacy of genes, which we had previously found to be differentially expressed in an in vitro model of human lung carcinogenesis.Experimental Design: Prediction algorithms and risk-score models were applied to the expression of the genes in publicly available NSCLC expression data sets. The prognostic capacity of the immunohistochemical expression of proteins encoded by these genes was also tested using formalin-fixed paraffin-embedded (FFPE) tissue specimens from 156 lung adenocarcinomas and 79 squamous cell carcinomas (SCCs).Results: The survival of all-stages (P P in vitro lung carcinogenesis model (FILM) signature was significantly poorer than that of patients who did not. No survival differences were observed between SCCs predicted to express or lack FILM signature. Moreover, all stages (P P = 0.001, HR = 2.6) adenocarcinoma patients predicted to be at high risk by FILM transcript exhibited significantly worse survival than patients at low risk. Furthermore, the corresponding protein signature was associated with poor survival (all stages, P < 0.001, HR = 3.6; stage-I, P < 0.001, HR = 3.5; stage-IB, P < 0.001, HR = 4.6) and mortality risk (all stages, P = 0.001, HR = 4.0; stage-I, P = 0.01, HR = 3.4; stage-IB, P Conclusions: Our findings highlight a gene and corresponding protein signature with effective capacity for identification of stage-I lung adenocarcinoma patients with poor prognosis that are likely to benefit from adjuvant therapy. Clin Cancer Res; 17(6); 1490–501. ©2010 AACR.

Supplementary methods including detailed methods on specimen collection and evaluation; DNA and RNA isolation; deep DNA targeted sequencing and transcriptome sequencing; identification and validation of somatic mutations; and expression analysis.