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Neoadjuvant ipilimumab + nivolumab (Ipi+Nivo) and nivolumab + chemotherapy (Nivo+CT) induce greater pathologic response rates than CT alone in patients with operable non-small cell lung cancer (NSCLC). The impact of adding ipilimumab to neoadjuvant Nivo+CT is unknown. Here we report the results and correlates of two arms of the phase 2 platform NEOSTAR trial testing neoadjuvant Nivo+CT and Ipi+Nivo+CT with major pathologic response (MPR) as the primary endpoint. MPR rates were 32.1% (7/22, 80% confidence interval (CI) 18.7–43.1%) in the Nivo+CT arm and 50% (11/22, 80% CI 34.6–61.1%) in the Ipi+Nivo+CT arm; the primary endpoint was met in both arms. In patients without known tumor EGFR/ALK alterations, MPR rates were 41.2% (7/17) and 62.5% (10/16) in the Nivo+CT and Ipi+Nivo+CT groups, respectively. No new safety signals were observed in either arm. Single-cell sequencing and multi-platform immune profiling (exploratory endpoints) underscored immune cell populations and phenotypes, including effector memory CD8+ T, B and myeloid cells and markers of tertiary lymphoid structures, that were preferentially increased in the Ipi+Nivo+CT cohort. Baseline fecal microbiota in patients with MPR were enriched with beneficial taxa, such as Akkermansia, and displayed reduced abundance of pro-inflammatory and pathogenic microbes. Neoadjuvant Ipi+Nivo+CT enhances pathologic responses and warrants further study in operable NSCLC. (ClinicalTrials.gov registration: NCT03158129.)

Tumor-infiltrating B and plasma cells (TIB) are prevalent in lung adenocarcinoma (LUAD); however, they are poorly characterized. We performed paired single-cell RNA and B-cell receptor (BCR) sequencing of 16 early-stage LUADs and 47 matching multiregion normal tissues. By integrative analysis of ∼50,000 TIBs, we define 12 TIB subsets in the LUAD and adjacent normal ecosystems and demonstrate extensive remodeling of TIBs in LUADs. Memory B cells and plasma cells (PC) were highly enriched in tumor tissues with more differentiated states and increased frequencies of somatic hypermutation. Smokers exhibited markedly elevated PCs and PCs with distinct differentiation trajectories. BCR clonotype diversity increased but clonality decreased in LUADs, smokers, and with increasing pathologic stage. TIBs were mostly localized within CXCL13+ lymphoid aggregates, and immune cell sources of CXCL13 production evolved with LUAD progression and included elevated fractions of CD4 regulatory T cells. This study provides a spatial landscape of TIBs in early-stage LUAD.Significance:While TIBs are highly enriched in LUADs, they are poorly characterized. This study provides a much-needed understanding of the transcriptional, clonotypic states and phenotypes of TIBs, unraveling their potential roles in the immunopathology of early-stage LUADs and constituting a road map for the development of TIB-targeted immunotherapies for the treatment of this morbid malignancy.This article is highlighted in the In This Issue feature, p. 2483

Supplementary Data from The Single-Cell Immunogenomic Landscape of B and Plasma Cells in Early-Stage Lung Adenocarcinoma

Supplementary Figure from The Single-Cell Immunogenomic Landscape of B and Plasma Cells in Early-Stage Lung Adenocarcinoma

Tumor-infiltrating B and plasma cells (TIB) are prevalent in lung adenocarcinoma (LUAD); however, they are poorly characterized. We performed paired single-cell RNA and B-cell receptor (BCR) sequencing of 16 early-stage LUADs and 47 matching multiregion normal tissues. By integrative analysis of ∼50,000 TIBs, we define 12 TIB subsets in the LUAD and adjacent normal ecosystems and demonstrate extensive remodeling of TIBs in LUADs. Memory B cells and plasma cells (PC) were highly enriched in tumor tissues with more differentiated states and increased frequencies of somatic hypermutation. Smokers exhibited markedly elevated PCs and PCs with distinct differentiation trajectories. BCR clonotype diversity increased but clonality decreased in LUADs, smokers, and with increasing pathologic stage. TIBs were mostly localized within CXCL13+ lymphoid aggregates, and immune cell sources of CXCL13 production evolved with LUAD progression and included elevated fractions of CD4 regulatory T cells. This study provides a spatial landscape of TIBs in early-stage LUAD.Significance:While TIBs are highly enriched in LUADs, they are poorly characterized. This study provides a much-needed understanding of the transcriptional, clonotypic states and phenotypes of TIBs, unraveling their potential roles in the immunopathology of early-stage LUADs and constituting a road map for the development of TIB-targeted immunotherapies for the treatment of this morbid malignancy.This article is highlighted in the In This Issue feature, p. 2483

Supplementary Fig S8 and legend

XLSX file - 627K, Supplementary table depicting 1395 genes significantly differentially expressed with time in the molecular field of injury

This file Includes 2 figures and one table as follow: 1: Supplementary figure S1 showing relative IL-22 mRNA expression (S1A), mutant K-ras protein expression (S1B), representative T regulatory cell flow cytometry plot (S1C), and total CD4 and CD8 flow cytometry plots (S1D) 2: Supplementary figure S2 showing pSTAT3 immunohistochemistry (S2A) and T cell related transcription factor mRNA expression (S2B) 3: Supplementary Table S1: List of Q-PCR primers and sequences

Supplementary Fig S9 and legend

Supplementary Figures S10-S20 and figure legends

Supplementary Methods. Supplementary Table 2. Details of the antibody used for TTF1 immunostaining.

Smoking perpetuates in cytologically normal airways a molecular “field of injury” that is pertinent to lung cancer and early detection. The evolution of airway field changes prior to lung oncogenesis is poorly understood largely due to the long latency of lung cancer in smokers. Here, we studied airway expression changes prior to lung cancer onset in mice with knockout of the Gprc5a gene (Gprc5a−/−) and tobacco carcinogen (NNK) exposure and that develop the most common type of lung cancer, lung adenocarcinoma, within 6 months following exposure. Airway epithelial brushings were collected from Gprc5a−/− mice before exposure and at multiple times post-NNK until time of lung adenocarcinoma development and then analyzed by RNA sequencing. Temporal airway profiles were identified by linear models and analyzed by comparative genomics in normal airways of human smokers with and without lung cancer. We identified significantly altered profiles (n = 926) in the NNK-exposed mouse normal airways relative to baseline epithelia, a subset of which were concordantly modulated with smoking status in the human airway. Among airway profiles that were significantly modulated following NNK, we found that expression changes (n = 22) occurring as early as 2 months following exposure were significantly associated with lung cancer status when examined in airways of human smokers. Furthermore, a subset of a recently reported human bronchial gene classifier (Percepta; n = 56) was enriched in the temporal mouse airway profiles. We underscore evolutionarily conserved profiles in the normal-appearing airway that develop prior to lung oncogenesis and that comprise viable markers for early lung cancer detection in suspect smokers. Cancer Prev Res; 11(4); 237–48. ©2018 AACR.

Perspective on this Article from Identification of Gene Signatures and Molecular Markers for Human Lung Cancer Prognosis using an In vitro Lung Carcinogenesis System

Somatic KRAS mutations are the most common oncogenic variants in lung cancer and are associated with poor prognosis. Using a Kras-induced lung cancer mouse model, CC-LR, we previously showed a role for inflammation in lung tumorigenesis through activation of the NF-κB pathway, along with induction of interleukin 6 (IL6) and an IL17-producing CD4+ T-helper cell response. IL22 is an effector molecule secreted by CD4+ and γδ T cells that we previously found to be expressed in CC-LR mice. IL22 mostly signals through the STAT3 pathway and is thought to act exclusively on nonhematopoietic cells with basal IL22 receptor (IL22R) expression on epithelial cells. Here, we found that higher expression of IL22R1 in patients with KRAS-mutant lung adenocarcinoma was an independent indicator of poor recurrence-free survival. We then showed that genetic ablation of Il22 in CC-LR mice (CC-LR/IL22KO mice) caused a significant reduction in tumor number and size. This was accompanied by significantly lower tumor cell proliferation, angiogenesis, and STAT3 activation. Il22 ablation was also associated with significant reduction in lung-infiltrating inflammatory cells and expression of protumor inflammatory cytokines. Conversely, this was accompanied with increased antitumor Th1 and cytotoxic CD8+ T-cell responses, while suppressing the protumor immunosuppressive T regulatory cell response. In CC-LR/IL22KO mice, we found significantly reduced expression of core stemness genes and the number of prototypical SPC+CCSP+ stem cells. Thus, we conclude that IL22 promotes Kras-mutant lung tumorigenesis by driving a protumor inflammatory microenvironment with proliferative, angiogenic, and stemness contextual cues in epithelial/tumor cells. Cancer Immunol Res; 6(7); 788–97. ©2018 AACR.

XLSX file - 102K, Supplementary table depicting 263 genes comprising a cluster of genes with highest average expression in adjacent airways

PDF file - 6272K, Sweave report depicting complete codes used for microarray analysis and identification of genes differentially expressed by site and time

Supplementary Figures S1-S7 and figure legends

Supplementary Figure from Chronic Exposure to Waterpipe Smoke Elicits Immunomodulatory and Carcinogenic Effects in the Lung

Supplementary Materials from Identification of Gene Signatures and Molecular Markers for Human Lung Cancer Prognosis using an In vitro Lung Carcinogenesis System

PDF file - 125K, Histograms of the p-values for site- and time-dependent differential expression; Site-dependent differential gene expression patterns identified by expression profiling of airways excluding main carinas; Smoothened scatter plot of transformed p-values for site and time effects

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

Supplementary Figures S21-S24 and 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

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.

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