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Very little is known about how the adaptive immune system responds to clonal evolution and tumor heterogeneity in non-small cell lung cancer. We profiled the T-cell receptor β complementarity determining region 3 in 20 patients with fully resected non-small cell lung cancer primary lesions and paired brain metastases. We characterized the richness, abundance and overlap of T cell clones between pairs, in addition to the tumor mutation burden and predicted neoantigens. We found a significant contraction in the number of unique T cell clones in brain metastases compared to paired primary cancers. The vast majority of T cell clones were specific to a single lesion, and there was minimal overlap in T cell clones between paired lesions. Despite the contraction in the number of T cell clones, brain metastases had higher non-synonymous mutation burdens than primary lesions. Our results suggest that there is greater richness of T cell clones in primary lung cancers than their paired metastases despite the higher mutation burden observed in metastatic lesions. These results may have implications for immunotherapy.

Introduction: We performed a genomic study in lung adenocarcinoma cases with discordant anaplastic lymphoma receptor tyrosine kinase gene (ALK) status by fluorescent in situ hybridization (FISH) and immunohistochemical (IHC) analysis., Methods: DNA from formalin-fixed paraffin-embedded tissues of 16 discordant (four FISH-positive/IHC-negative and 12 FISH-negative/IHC-positive) cases by Vysis ALK Break Apart FISH and ALK IHC testing (ALK1 clone) were subjected to whole gene capture and next-generation sequencing (NGS) of nine genes, including ALK, echinoderm microtubule associated protein like 4 gene (EML4), kinesin family member 5B gene (KIF5B), staphylococcal nuclease and tudor domain containing 1 gene (SND1), BRAF, ret proto-oncogene (RET), ezrin gene (EZR), ROS1, and telomerase reverse transcriptase (TERT). All discordant cases (except one FISH-negative/IHC-positive case without sufficient tissue) were analyzed by IHC with D5F3 antibody. In one case with fresh frozen tissue, whole transcriptome sequencing was also performed. Twenty-six concordant (16 FISH-positive/IHC-positive and 10 FISH-negative/IHC-negative) cases were included as controls., Results: In four ALK FISH-positive/IHC-negative cases, no EML4-ALK fusion gene was observed by NGS, but in one case using fresh frozen tissue, we identified EML4-baculoviral AIP repeat containing 6 gene (BIRC6) and AP2 associated kinase 1 gene (AAK1)-ALK fusion genes. Whole transcriptome sequencing revealed a highly expressed EML4-BIRC6 fusion transcript and a minimally expressed AAK1 transcript. Among the 12 FISH-negative/IHC-positive cases, no evidence of ALK gene rearrangement was detected by NGS. Eleven of 12 FISH-negative/IHC-positive cases detected by ALK1 clone were concordant by repeat ALK IHC with D5F3 antibody (i.e., FISH-negative/IHC-negative by D5F3 clone). Among the 16 ALK FISH-positive/IHC-positive positive controls, whole gene capture identified ALK gene fusion in 15 cases, including in one case with Huntington interacting protein 1 gene (HIP1)-ALK. No ALK fusion gene was observed in any of the 10 FISH-negative/IHC-negative cases. Other fusion genes involving ROS1, EZR, BRAF, and SND1 were also found., Conclusions: ALK FISH results appeared to be false-positive in three of four FISH-positive/IHC-negative cases, whereas no false-negative ALK FISH case was identified among 12 ALK FISH-negative/IHC-positive cases by ALK1 clone, which was in keeping with the concordant FISH-negative/IHC-negative status by D5F3 clone. Our targeted whole gene capture approach using formalin-fixed paraffin embedded samples was effective for detecting rearrangements involving ALK and other actionable oncogenes., (Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Lung adenocarcinomas from never smokers account for approximately 15 to 20% of all lung cancers and these tumors often carry genetic alterations that are responsive to targeted therapy. Here we examined mutation status in 10 oncogenes among 89 lung adenocarcinomas from never smokers. We also screened for oncogene fusion transcripts in 20 of the 89 tumors by RNA-Seq. In total, 62 tumors had mutations in at least one of the 10 oncogenes, including EGFR (49 cases, 55%), K-ras (5 cases, 6%), BRAF (4 cases, 5%), PIK3CA (3 cases, 3%), and ERBB2 (4 cases, 5%). In addition to ALK fusions identified by IHC/FISH in four cases, two previously known fusions involving EZR- ROS1 and KIF5B-RET were identified by RNA-Seq as well as a third novel fusion transcript that was formed between exons 1-9 of SND1 and exons 2 to 3' end of BRAF. This in-frame fusion was observed in 3/89 tested tumors and 2/64 additional never smoker lung adenocarcinoma samples. Ectopic expression of SND1-BRAF in H1299 cells increased phosphorylation levels of MEK/ERK, cell proliferation, and spheroid formation compared to parental mock-transfected control. Jointly, our results suggest a potential role of the novel BRAF fusion in lung cancer development and therapy.

Lung cancer occurs in never-smokers. Epigenetic changes in lung cancer potentially represent important diagnostic, prognostic, and therapeutic targets. We compared DNA methylation profiles of 28 adenocarcinomas of the lungs of never-smokers with paired adjacent nonmalignant lung tissue. We correlated differential methylation changes with gene expression changes from the same 28 sample pairs. Using principal component analysis, we observed a distinct separation in methylation profiles between tumor and adjacent nonmalignant lung tissue. Tumors were generally hypomethylated compared with adjacent nonmalignant tissue. Of 1,906 CpG sites differentially methylated between tumor and nonmalignant tissue, 1,198 were within classically defined CpG islands where tumors were hypermethylated compared with nonmalignant tissue. A total of 708 sites were outside CpG islands where tumors were hypomethylated compared with nonmalignant tissue. There were significant differences in expression of 351 genes (23%) of the 1,522 genes matched to the differentially methylated CpG sites. Genes that were not significantly differentially expressed and were hypermethylated within CpG sites were enriched for homeobox genes. These results suggest that the methylation profiles of lung adenocarcinomas of never-smokers and adjacent nonmalignant lung tissue are significantly different. Despite the differential methylation of homeobox genes, no significant changes in expression of these genes were detected., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Novel and targetable mutations are needed for improved understanding and treatment of lung cancer in never-smokers., Methods: Twenty-seven lung adenocarcinomas from never-smokers were sequenced by both exome and mRNA-seq with respective normal tissues. Somatic mutations were detected and compared with pathway deregulation, tumor phenotypes and clinical outcomes., Results: Although somatic mutations in DNA or mRNA ranged from hundreds to thousands in each tumor, the overlap mutations between the two were only a few to a couple of hundreds. The number of somatic mutations from either DNA or mRNA was not significantly associated with clinical variables; however, the number of overlap mutations was associated with cancer subtype. These overlap mutants were preferentially expressed in mRNA with consistently higher allele frequency in mRNA than in DNA. Ten genes (EGFR, TP53, KRAS, RPS6KB2, ATXN2, DHX9, PTPN13, SP1, SPTAN1 and MYOF) had recurrent mutations and these mutations were highly correlated with pathway deregulation and patient survival., Conclusions: The recurrent mutations present in both DNA and RNA are likely the driver for tumor biology, pathway deregulation and clinical outcomes. The information may be used for patient stratification and therapeutic target development.

Objective: This study was designed to determine the prognostic value of BCAR1 expression and its associations with clinical-demographical characteristics in multiple centers of non-small cell lung cancer (NSCLC) patients., Methods: Gene expression microarray (mRNA) of 77 adenocarcinomas from Mayo Clinic, RNA-sequencing of 508 NSCLC from The Cancer Genome Atlas (TCGA), and immunohistochemistry stain of BCAR1-protein expression in 150 cases from Daping Hospital were included in the study. The association of mRNA or protein expression with patient clinical characteristics and overall survival was assessed in each dataset. We also predicted microRNAs (miRNA) that target BCAR1 using bioinformatics prediction tools and evaluated miRNA expression patterns with BCAR1 expression in miRNA-sequencing data of 74 lung cancer cases from TCGA dataset., Results: In the Mayo Clinic dataset, a higher BCAR1-mRNA level correlated significantly with more advanced tumor-stage and lymphatic metastasis. Similar changes were observed in the TCGA RNA-seq dataset. Additionally, higher BCAR1-mRNA levels predicted poorer survival in adenocarcinoma and squamous carcinoma from the TCGA dataset. The protein levels in the adenocarcinoma cases with lymphatic metastasis were significantly higher than of those without metastasis. Tumor tissues demonstrated remarkably higher levels of protein compared with matched normal tissues although there was no significant difference in BCAR1-mRNA expression between tumor and matched normal tissues was detected. In miRNAs that were downregulated in the tumors, Let-7f-2 and miR-22 differed the most (P < 0.001 and P = 0.007, respectively)., Conclusions: We confirmed that increased BCAR1 expression predicts poorer prognosis in NSCLC. We postulate that mRNA-protein decoupling of BCAR1 may be a result of reduced inhibition of specific miRNAs in tumor tissues, which warrants further study.

Lung adenocarcinoma is the most common type of primary lung cancer. The purpose of this study was to delineate gene expression patterns for survival prediction in lung adenocarcinoma. Gene expression profiles of 82 (discovery set) and 442 (validation set 1) lung adenocarcinoma tumor tissues were analyzed using a systems biology-based network approach. We also examined the expression profiles of 78 adjacent normal lung tissues from 82 patients. We found a significant correlation of an expression module with overall survival (adjusted hazard ratio or HR=1.71; 95% CI=1.06-2.74 in discovery set; adjusted HR=1.26; 95% CI=1.08-1.49 in validation set 1). This expression module contained genes enriched in the biological process of the cell cycle. Interestingly, the cell cycle gene module and overall survival association were also significant in normal lung tissues (adjusted HR=1.91; 95% CI, 1.32-2.75). From these survival-related modules, we further defined three hub genes (UBE2C, TPX2, and MELK) whose expression-based risk indices were more strongly associated with poor 5-year survival (HR=3.85, 95% CI=1.34-11.05 in discovery set; HR=1.72, 95% CI=1.21-2.46 in validation set 1; and HR=3.35, 95% CI=1.08-10.04 in normal lung set). The 3-gene prognostic result was further validated using 92 adenocarcinoma tumor samples (validation set 2); patients with a high-risk gene signature have a 1.52-fold increased risk (95% CI, 1.02-2.24) of death than patients with a low-risk gene signature. These results suggest that a network-based approach may facilitate discovery of key genes that are closely linked to survival in patients with lung adenocarcinoma., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Purpose: miRNA plays an important role in human disease and cancer. We seek to investigate the expression status, clinical relevance, and functional role of miRNA in non-small cell lung cancer., Experimental Design: We conducted miRNA expression profiling in matched lung adenocarcinoma and uninvolved lung using 56 pairs of fresh-frozen (FF) and 47 pairs of formalin-fixed, paraffin-embedded (FFPE) samples from never smokers. The most differentially expressed miRNA genes were evaluated by Cox analysis and log-rank test. Among the best candidate, miR-708 was further examined for differential expression in two independent cohorts. Functional significance of miR-708 expression in lung cancer was examined by identifying its candidate mRNA target and through manipulating its expression levels in cultured cells., Results: Among the 20 miRNAs most differentially expressed between tested tumor and normal samples, high expression level of miR-708 in the tumors was most strongly associated with an increased risk of death after adjustments for all clinically significant factors including age, sex, and tumor stage (FF cohort: HR, 1.90; 95% CI, 1.08-3.35; P = 0.025 and FFPE cohort: HR, 1.93; 95% CI, 1.02-3.63; P = 0.042). The transcript for TMEM88 gene has a miR-708 binding site in its 3' UTR and was significantly reduced in tumors high of miR-708. Forced miR-708 expression reduced TMEM88 transcript levels and increased the rate of cell proliferation, invasion, and migration in culture., Conclusions: miRNA-708 acts as an oncogene contributing to tumor growth and disease progression by directly downregulating TMEM88, a negative regulator of the Wnt signaling pathway in lung cancer., (©2012 AACR.)

Chronic obstructive pulmonary disease (COPD) is a strong risk factor for lung cancer. Published studies about variations of genes encoding glutathione metabolism, DNA repair, and inflammatory response pathways in susceptibility to COPD were inconclusive. We evaluated 470 single-nucleotide polymorphisms (SNP) from 56 genes of these three pathways in 620 cases and 893 controls to identify susceptibility markers for COPD risk, using existing resources. We assessed SNP- and gene-level effects adjusting for sex, age, and smoking status. Differential genetic effects on disease risk with and without lung cancer were also assessed; cumulative risk models were established. Twenty-one SNPs were found to be significantly associated with risk of COPD (P < 0.01); gene-based analyses confirmed two genes (GCLC and GSS) and identified three additional genes (GSTO2, ERCC1, and RRM1). Carrying 12 high-risk alleles may increase risk by 2.7-fold; eight SNPs altered COPD risk without lung cancer by 3.1-fold and 4 SNPs altered the risk with lung cancer by 2.3-fold. Our findings indicate that multiple genetic variations in the three selected pathways contribute to COPD risk through GCLC, GSS, GSTO2, ERCC1, and RRM1 genes. Functional studies are needed to elucidate the mechanisms of these genes in the development of COPD, lung cancer, or both.

Background: Genetic susceptibility in lung cancer risk has long been recognized but remains ill defined, as does the role of tobacco smoke exposure and chronic obstructive pulmonary disease (COPD)., Methods: Using a dual case-control design, we tested whether alpha(1)-antitrypsin deficiency (alpha(1)ATD) carriers are predisposed to a higher risk of lung cancer, adjusting for the effects of tobacco smoke exposure and COPD. A total of 1856 patients with incident lung cancer were included in the study; 1585 community residents served as controls. A second control group was composed of 902 full siblings of the patients. We first modeled 1585 case-control pairs without the alpha(1)ATD variable using multiple logistic regression analysis and then modeled the alpha(1)ATD allele type in the presence of other known risk factors of lung cancer., Results: We found a significantly increased lung cancer risk among alpha(1)ATD carriers from 2 parallel case-control comparisons: when patients were compared with unrelated controls, alpha(1)ATD carriers had a 70% higher risk of developing lung cancer than noncarriers (odds ratio, 1.7; 95% confidence interval, 1.2-2.4). In a further comparison of patients with their cancer-free siblings, we found a 2-fold increased lung cancer risk in alpha(1)ATD carriers (95% confidence interval, 1.4-2.7). Stratified analysis by tumor histologic subtypes showed a significant increase for adenocarcinoma and squamous cell carcinoma among alpha(1)ATD carriers., Conclusion: Our results suggest that alpha(1)ATD carriers are at a 70% to 100% increased risk of lung cancer and may account for 11% to 12% of the patients with lung cancer in our study.

Purpose: Gene expression profiling for outcome prediction of non-small-cell lung cancer (NSCLC) remains clouded by heterogeneous and unvalidated results. This study applied multivariate approaches to identify and evaluate value-added gene expression signatures in two types of NSCLC., Materials and Methods: Two NSCLC oligonucleotide microarray data sets of adenocarcinoma and squamous cell carcinoma were used as training sets to select prognostic genes independent of conventional predictors. The top 50 genes from each set were used to predict the outcomes of two independent validation data sets of 84 and 91 NSCLC cases., Results: Adenocarcinomas with the 50-gene signature from adenocarcinoma in both validation data sets had a 2.4-fold (95% CI, 1.3 to 4.4 and 1.0 to 5.8) increased mortality after adjustment for conventional predictors. Squamous cell carcinoma with this high-risk signature had an adjusted risk of 1.1 (95% CI, 0.4 to 3.2) in one data set and 2.5 (95% CI, 1.1 to 5.8) in another consisting of stage I tumors. Adenocarcinoma with the 50-gene signature from squamous cell carcinoma had an elevated risk of 3.5 (95% CI, 1.4 to 9.0) after adjustment for conventional predictors. Squamous cell carcinoma with this high risk signature had an adjusted risk of 1.8 (95% CI, 0.7 to 4.6). Despite the little overlap in individual genes, the two gene signatures had significant functional connectedness in molecular pathways., Conclusion: Two non-overlapping but functionally related gene expression signatures provide consistently improved survival prediction for NSCLC regardless of histologic cell type. Multiple sets of genes may exist for NSCLC with predictive value, but ones with independent predictive value beyond clinical predictors will be required for clinical translation.