Comprehensive molecular profiling of lung adenocarcinoma. Nature Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis. 10.1038/nature13385
    Germline mutations in young non-smoking women with lung adenocarcinoma. Donner Iikki,Katainen Riku,Sipilä Lauri J,Aavikko Mervi,Pukkala Eero,Aaltonen Lauri A Lung cancer (Amsterdam, Netherlands) OBJECTIVES:Although the primary cause of lung cancer is smoking, a considerable proportion of all lung cancers occur in never smokers. Gender influences the risk and characteristics of lung cancer and women are overrepresented among never smokers with the disease. Young age at onset and lack of established environmental risk factors suggest genetic predisposition. In this study, we used population-based sampling of young patients to discover candidate predisposition variants for lung adenocarcinoma in never-smoking women. MATERIALS AND METHODS:We employed archival normal tissue material from 21 never-smoker women who had been diagnosed with lung adenocarcinoma before the age of 45, and exome sequenced their germline DNA. RESULTS AND CONCLUSION:Potentially pathogenic variants were found in eight Cancer Gene Census germline genes: BRCA1, BRCA2, ERCC4, EXT1, HNF1 A, PTCH1, SMARCB1 and TP53. The variants in TP53, BRCA1, and BRCA2 are likely to have contributed to the early onset lung cancer in the respective patients (3/21 or 14%). This supports the notion that lung adenocarcinoma can be a component of certain cancer predisposition syndromes. Fifteen genes displayed potentially pathogenic mutations in at least two patients: ABCC10, ATP7B, CACNA1S, CFTR, CLIP4, COL6A1, COL6A6, GCN1, GJB6, RYR1, SCN7A, SEC24A, SP100, TTN and USH2A. Four patients showed a mutation in COL6A1, three in CLIP4 and two in the rest of the genes. Some of these candidate genes may explain a subset of female lung adenocarcinoma. 10.1016/j.lungcan.2018.05.027
    Development of Kras mutant lung adenocarcinoma in mice with knockout of the airway lineage-specific gene Gprc5a. Fujimoto Junya,Nunomura-Nakamura Sayuri,Liu Yihua,Lang Wenhua,McDowell Tina,Jakubek Yasminka,Ezzeddine Dalia,Kapere Ochieng Joshua,Petersen Jason,Davies Gareth,Fukuoka Junya,Wistuba Ignacio I,Ehli Erik,Fowler Jerry,Scheet Paul,Kadara Humam International journal of cancer Despite the urgency for prevention and treatment of lung adenocarcinoma (LUAD), we still do not know drivers in pathogenesis of the disease. Earlier work revealed that mice with knockout of the G-protein coupled receptor Gprc5a develop late onset lung tumors including LUADs. Here, we sought to further probe the impact of Gprc5a expression on LUAD pathogenesis. We first surveyed GPRC5A expression in human tissues and found that GPRC5A was markedly elevated in human normal lung relative to other normal tissues and was consistently downregulated in LUADs. In sharp contrast to wild-type littermates, Gprc5a mice treated chronically with the nicotine-specific carcinogen NNK developed LUADs by 6 months following NNK exposure. Immunofluorescence analysis revealed that the LUADs exhibited abundant expression of surfactant protein C and lacked the clara cell marker Ccsp, suggesting that these LUADs originated from alveolar type II cells. Next, we sought to survey genome-wide alterations in the pathogenesis of Gprc5a LUADs. Using whole exome sequencing, we found that carcinogen-induced LUADs exhibited markedly higher somatic mutation burdens relative to spontaneous tumors. All LUADs were found to harbor somatic mutations in the Kras oncogene (p. G12D or p. Q61R). In contrast to spontaneous lesions, carcinogen-induced Gprc5a LUADs exhibited mutations (variants and copy number gains) in additional drivers (Atm, Kmt2d, Nf1, Trp53, Met, Ezh2). Our study underscores genomic alterations that represent early events in the development of Kras mutant LUAD following Gprc5a loss and tobacco carcinogen exposure and that may constitute targets for prevention and early treatment of this disease. 10.1002/ijc.30851
    Transcriptome Sequencing Reveals Key Pathways and Genes Associated with Cisplatin Resistance in Lung Adenocarcinoma A549 Cells. Fang Yani,Zhang Cheng,Wu Tong,Wang Qi,Liu Jinhui,Dai Penggao PloS one Acquired resistance to cisplatin-based chemotherapy frequently occurs in patients with non-small cell lung cancer, and the underlying molecular mechanisms are not well understood. The aim of this study was to investigate whether a distinct gene expression pattern is associated with acquired resistance to cisplatin in human lung adenocarcinoma. Whole-transcriptome sequencing was performed to compare the genome-wide gene expression patterns of the human lung adenocarcinoma A549 cisplatin-resistant cell line A549/DDP with those of its progenitor cell line A549. A total of 1214 differentially expressed genes (DEGs) were identified, 656 of which were upregulated and 558 were downregulated. Functional annotation of the DEGs in the Kyoto Encyclopedia of Genes and Genomes database revealed that most of the identified genes were enriched in the PI3K/AKT, mitogen-activated protein kinase, actin cytoskeleton regulation, and focal adhesion pathways in A549/DDP cells. These results support previous studies demonstrating that the pathways regulating cell proliferation and invasion confer resistance to chemotherapy. Furthermore, the results proved that cell adhesion and cytoskeleton regulation is associated with cisplatin resistance in human lung cancer. Our study provides new promising biomarkers for lung cancer prognosis and potential therapeutic targets for lung cancer treatment. 10.1371/journal.pone.0170609
    Carcinogen-induced tumors in SFN-transgenic mice harbor a characteristic mutation spectrum of human lung adenocarcinoma. Kim Yunjung,Shiba-Ishii Aya,Ramirez Karina,Muratani Masafumi,Sakamoto Noriaki,Iijima Tatsuo,Noguchi Masayuki Cancer science The landscape of genetic alterations in disease models such as transgenic mice or mice with carcinogen-induced tumors has provided a huge amount of information that has shed light on the process of tumorigenesis in human non-small-cell lung cancer (NSCLC). We have previously identified stratifin (SFN) as a potent oncogene, and generated SFN-transgenic (Tg-SPC-SFN ) mice, which express human SFN (hSFN) only in the lung. Here, we have found that carcinogen nicotine-derived nitrosaminoketone (NNK)-induced tumors developing in Tg-SPC-SFN mice show a similar histology to human lung adenocarcinoma and exhibit high hSFN expression. In order to compare the genetic characteristics of Tg-SPC-SFN tumors and human lung adenocarcinoma, the former were subjected to whole-exome sequencing. Interestingly, Tg-SPC-SFN tumors showed the distinct distribution of exonic mutations and high number of mutated genes and transversion. Moreover, Tg-SPC-SFN tumors showed 73 genes that were commonly detected in more than 2 tumors, mutations of which were also found in human lung adenocarcinoma. The expression levels of some of these genes were significantly associated with the clinical outcome of lung adenocarcinoma patients. Additionally, mutated genes in Tg-SPC-SFN tumors were closely associated with key canonical pathways such as PI3K/AKT signaling and apoptosis signaling. These results suggest that SFN overexpression is a universal abnormality in human lung adenocarcinogenesis and Tg-SPC-SFN tumors recapitulate key features of major human lung adenocarcinoma. Therefore, Tg-SPC-SFN mice provide a useful model for clarifying the molecular mechanism underlying lung adenocarcinogenesis. 10.1111/cas.14081
    Genomic and immune profiling of pre-invasive lung adenocarcinoma. Chen Haiquan,Carrot-Zhang Jian,Zhao Yue,Hu Haichuan,Freeman Samuel S,Yu Su,Ha Gavin,Taylor Alison M,Berger Ashton C,Westlake Lindsay,Zheng Yuanting,Zhang Jiyang,Ramachandran Aruna,Zheng Qiang,Pan Yunjian,Zheng Difan,Zheng Shanbo,Cheng Chao,Kuang Muyu,Zhou Xiaoyan,Zhang Yang,Li Hang,Ye Ting,Ma Yuan,Gao Zhendong,Tao Xiaoting,Han Han,Shang Jun,Yu Ying,Bao Ding,Huang Yechao,Li Xiangnan,Zhang Yawei,Xiang Jiaqing,Sun Yihua,Li Yuan,Cherniack Andrew D,Campbell Joshua D,Shi Leming,Meyerson Matthew Nature communications Adenocarcinoma in situ and minimally invasive adenocarcinoma are the pre-invasive forms of lung adenocarcinoma. The genomic and immune profiles of these lesions are poorly understood. Here we report exome and transcriptome sequencing of 98 lung adenocarcinoma precursor lesions and 99 invasive adenocarcinomas. We have identified EGFR, RBM10, BRAF, ERBB2, TP53, KRAS, MAP2K1 and MET as significantly mutated genes in the pre/minimally invasive group. Classes of genome alterations that increase in frequency during the progression to malignancy are revealed. These include mutations in TP53, arm-level copy number alterations, and HLA loss of heterozygosity. Immune infiltration is correlated with copy number alterations of chromosome arm 6p, suggesting a link between arm-level events and the tumor immune environment. 10.1038/s41467-019-13460-3
    Somatic Genomics and Clinical Features of Lung Adenocarcinoma: A Retrospective Study. Shi Jianxin,Hua Xing,Zhu Bin,Ravichandran Sarangan,Wang Mingyi,Nguyen Cu,Brodie Seth A,Palleschi Alessandro,Alloisio Marco,Pariscenti Gianluca,Jones Kristine,Zhou Weiyin,Bouk Aaron J,Boland Joseph,Hicks Belynda,Risch Adam,Bennett Hunter,Luke Brian T,Song Lei,Duan Jubao,Liu Pengyuan,Kohno Takashi,Chen Qingrong,Meerzaman Daoud,Marconett Crystal,Laird-Offringa Ite,Mills Ian,Caporaso Neil E,Gail Mitchell H,Pesatori Angela C,Consonni Dario,Bertazzi Pier Alberto,Chanock Stephen J,Landi Maria Teresa PLoS medicine BACKGROUND:Lung adenocarcinoma (LUAD) is the most common histologic subtype of lung cancer and has a high risk of distant metastasis at every disease stage. We aimed to characterize the genomic landscape of LUAD and identify mutation signatures associated with tumor progression. METHODS AND FINDINGS:We performed an integrative genomic analysis, incorporating whole exome sequencing (WES), determination of DNA copy number and DNA methylation, and transcriptome sequencing for 101 LUAD samples from the Environment And Genetics in Lung cancer Etiology (EAGLE) study. We detected driver genes by testing whether the nonsynonymous mutation rate was significantly higher than the background mutation rate and replicated our findings in public datasets with 724 samples. We performed subclonality analysis for mutations based on mutant allele data and copy number alteration data. We also tested the association between mutation signatures and clinical outcomes, including distant metastasis, survival, and tumor grade. We identified and replicated two novel candidate driver genes, POU class 4 homeobox 2 (POU4F2) (mutated in 9 [8.9%] samples) and ZKSCAN1 (mutated in 6 [5.9%] samples), and characterized their major deleterious mutations. ZKSCAN1 was part of a mutually exclusive gene set that included the RTK/RAS/RAF pathway genes BRAF, EGFR, KRAS, MET, and NF1, indicating an important driver role for this gene. Moreover, we observed strong associations between methylation in specific genomic regions and somatic mutation patterns. In the tumor evolution analysis, four driver genes had a significantly lower fraction of subclonal mutations (FSM), including TP53 (p = 0.007), KEAP1 (p = 0.012), STK11 (p = 0.0076), and EGFR (p = 0.0078), suggesting a tumor initiation role for these genes. Subclonal mutations were significantly enriched in APOBEC-related signatures (p < 2.5×10-50). The total number of somatic mutations (p = 0.0039) and the fraction of transitions (p = 5.5×10-4) were associated with increased risk of distant metastasis. Our study's limitations include a small number of LUAD patients for subgroup analyses and a single-sample design for investigation of subclonality. CONCLUSIONS:These data provide a genomic characterization of LUAD pathogenesis and progression. The distinct clonal and subclonal mutation signatures suggest possible diverse carcinogenesis pathways for endogenous and exogenous exposures, and may serve as a foundation for more effective treatments for this lethal disease. LUAD's high heterogeneity emphasizes the need to further study this tumor type and to associate genomic findings with clinical outcomes. 10.1371/journal.pmed.1002162
    Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma with fully annotated clinical follow-up. Kadara H,Choi M,Zhang J,Parra E R,Rodriguez-Canales J,Gaffney S G,Zhao Z,Behrens C,Fujimoto J,Chow C,Yoo Y,Kalhor N,Moran C,Rimm D,Swisher S,Gibbons D L,Heymach J,Kaftan E,Townsend J P,Lynch T J,Schlessinger J,Lee J,Lifton R P,Wistuba I I,Herbst R S Annals of oncology : official journal of the European Society for Medical Oncology Background:Lung adenocarcinomas (LUADs) lead to the majority of deaths attributable to lung cancer. We performed whole-exome sequencing (WES) and immune profiling analyses of a unique set of clinically annotated early-stage LUADs to better understand the pathogenesis of this disease and identify clinically relevant molecular markers. Methods:We performed WES of 108 paired stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Ten immune markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry (IHC) in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fisher's exact test. Cox proportional hazards regression models were used for multivariate analysis of clinical outcome. Results:LUADs in this cohort exhibited an average of 243 coding mutations. We identified 28 genes with significant enrichment for mutation. SETD2-mutated LUADs exhibited relatively poor recurrence- free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS among KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis revealed that LUADs in smokers and with relatively high mutation burdens exhibited increased levels of immune markers. Analysis of immunophenotypes revealed that LUADs with STK11 mutations exhibited relatively low levels of infiltrating CD4+/CD8+ T-cells indicative of a muted immune response. Tumoral PD-L1 was significantly elevated in TP53 mutant LUADs whereas PIK3CA mutant LUADs exhibited markedly down-regulated PD-L1 expression. LUADs with TP53 or KEAP1 mutations displayed relatively increased CD57 and Granzyme B levels indicative of augmented natural killer (NK) cell infiltration. Conclusion(s):Our study highlights molecular and immune phenotypes that warrant further analysis for their roles in clinical outcomes and personalized immune-based therapy of LUAD. 10.1093/annonc/mdw436
    Whole-exome sequencing identifies key mutated genes in T790M wildtype/cMET-unamplified lung adenocarcinoma with acquired resistance to first-generation EGFR tyrosine kinase inhibitors. Li Chenguang,Liu Hailin,Zhang Bin,Gong Liqun,Su Yanjun,Zhang Zhenfa,Wang Changli Journal of cancer research and clinical oncology PURPOSE:Lung cancer is the leading cause of cancer-related death worldwide. Lung adenocarcinoma harboring EGFR-activating mutations will inevitably acquire resistance to first-generation EGFR tyrosine kinase inhibitors (TKIs). EGFR T790M mutation and cMET amplification are common mechanisms. Further study is needed to explore unknown genomic alterations contributing to drug resistance. METHODS:Tumor and blood samples from 69 stage IIIB-IV NSCLC patients defined as acquired resistance to first-generation EGFR TKIs (gefitinib, erlotinib or ecotinib) were collected. The cobas and Droplet digital PCR (ddPCR) were used to detect T790M mutations in tumor samples and plasma ctDNA. cMET amplification was evaluated by fluorescence in situ hybridization (FISH). Exome sequencing was performed in four T790M wildtype/cMET-unamplified samples. RESULTS:The overall T790M-positive rate was 52.2% considering all testing methods. Out of 21 samples in which tumor re-biopsy was performed, 14 were T790M positive (66.7%). cMET amplification was identified in three out of seven T790M-negative samples. Exome sequencing in four T790M wildtype/cMET-unamplified samples and paired white blood cells identified a cohort of candidate key mutated genes including BRAF, FGFR1, PAK1, PCNT, PEBP4 and SOX3. CONCLUSIONS:EGFR T790M mutation and cMET amplification are main mechanisms leading to EGFR TKI resistance in lung adenocarcinoma. These key mutated genes identified in the present study would need further validation in large number of patients. 10.1007/s00432-018-2634-4
    Tumor immune microenvironment and nivolumab efficacy in EGFR mutation-positive non-small-cell lung cancer based on T790M status after disease progression during EGFR-TKI treatment. Haratani K,Hayashi H,Tanaka T,Kaneda H,Togashi Y,Sakai K,Hayashi K,Tomida S,Chiba Y,Yonesaka K,Nonagase Y,Takahama T,Tanizaki J,Tanaka K,Yoshida T,Tanimura K,Takeda M,Yoshioka H,Ishida T,Mitsudomi T,Nishio K,Nakagawa K Annals of oncology : official journal of the European Society for Medical Oncology Background:The efficacy of programmed death-1 blockade in epidermal growth factor receptor gene (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) patients with different mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) is unknown. We retrospectively evaluated nivolumab efficacy and immune-related factors in such patients according to their status for the T790M resistance mutation of EGFR. Patients and methods:We identified 25 patients with EGFR mutation-positive NSCLC who were treated with nivolumab after disease progression during EGFR-TKI treatment (cohort A). Programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) density in tumor specimens obtained after acquisition of EGFR-TKI resistance were determined by immunohistochemistry. Whole-exome sequencing of tumor DNA was carried out to identify gene alterations. The relation of T790M status to PD-L1 expression or TIL density was also examined in an independent cohort of 60 patients (cohort B). Results:In cohort A, median progression-free survival (PFS) was 2.1 and 1.3 months for T790M-negative and T790M-positive patients, respectively (P = 0.099; hazard ratio of 0.48 with a 95% confidence interval of 0.20-1.24). Median PFS was 2.1 and 1.3 months for patients with a PD-L1 expression level of ≥1% or <1%, respectively (P = 0.084; hazard ratio of 0.37, 95% confidence interval of 0.10-1.21). PFS tended to increase as the PD-L1 expression level increased with cutoff values of ≥10% and ≥50%. The proportion of tumors with a PD-L1 level of ≥10% or ≥50% was higher among T790M-negative patients than among T790M-positive patients of both cohorts A and B. Nivolumab responders had a significantly higher CD8+ TIL density and nonsynonymous mutation burden. Conclusion:T790M-negative patients with EGFR mutation-positive NSCLC are more likely to benefit from nivolumab after EGFR-TKI treatment, possibly as a result of a higher PD-L1 expression level, than are T790M-positive patients. 10.1093/annonc/mdx183
    Molecular profiling of key driver genes improves staging accuracy in multifocal non-small cell lung cancer. Zheng Richard,Shen Qian,Mardekian Stacey,Solomides Charalambos,Wang Zi-Xuan,Evans Nathaniel R The Journal of thoracic and cardiovascular surgery OBJECTIVE:Multifocal non-small cell lung cancer has historically been separated into synchronous primary lung cancers or intrapulmonary metastases with the use of histopathology. We hypothesize that using targeted next-generation sequencing of key driver mutations in multifocal non-small cell lung cancer will improve our ability to differentiate intrapulmonary metastases from synchronous primary lung cancers. METHODS:We identified patients who underwent surgery for non-small cell lung cancer between 2013 and 2018 with multifocal tumors. Archived specimens were reviewed with a 4-gene next-generation sequencing panel identifying mutations of EGFR, KRAS, BRAF, and NRAS. Synchronous primary lung cancers were classified as lesions with different histopathologic subtypes or driver mutations. Tests of hypotheses were performed with the Fisher exact test. Calculations were performed in Stata (v13.0; StataCorp LLC, College Station, Tex). RESULTS:A total of 18 patients had non-small cell lung cancer tumor specimens (n = 41) available from 2 or more sites. The pathologic diagnosis was predominantly adenocarcinoma (39/41 specimens). We detected a driver mutation in 68.3% (28/41) of all tumors. The most common mutations observed were in KRAS (n = 17/41) and EGFR (n = 7/41). Eleven patients had synchronous primary lung cancers, and 4 patients had intrapulmonary metastases based on combined histopathologic and molecular profiling results. Three lacked driver mutations in either lesion. Eight synchronous primary lung cancers (8/18, 44%) were downstaged when compared with their original diagnosis (P = .08). Of these, 4 patients received adjuvant chemotherapy unnecessarily in hindsight. CONCLUSIONS:Molecular non-small cell lung cancer profiling using a 4-gene next-generation sequencing panel allows for better distinction between synchronous primary lung cancers and intrapulmonary metastases than histopathology alone. Routine use of next-generation sequencing for multifocal lesions prevents unnecessary adjuvant treatment for patients with histologically similar synchronous primary lung cancers. 10.1016/j.jtcvs.2019.11.126
    Transcriptome-based molecular subtyping of non-small cell lung cancer may predict response to immune checkpoint inhibitors. Jang Hee-Jin,Lee Hyun-Sung,Ramos Daniela,Park In Kyu,Kang Chang Hyun,Burt Bryan M,Kim Young Tae The Journal of thoracic and cardiovascular surgery OBJECTIVES:We set out to investigate whether transcriptome-based molecular subtypes in lung adenocarcinoma and lung squamous cell carcinoma are predictive of the response to programmed cell death 1 blockade. METHODS:Molecular classification of non-small cell lung cancer was performed by unsupervised clustering of mRNA sequencing data from 87 lung adenocarcinoma and 101 lung squamous cell carcinoma specimens, and molecular subtypes were characterized according to their immunogenomic determinants. A prediction algorithm of molecular subtypes was applied to 35 patients with non-small cell lung cancer treated with programmed cell death 1 blockade to test its association with treatment response (GSE93157; the Barcelona cohort). RESULTS:Unsupervised hierarchical clustering of transcriptome sequencing data in lung adenocarcinoma and lung squamous cell carcinoma revealed 3 and 2 distinct clusters, respectively. Cluster 1 in each histology had a higher expression of immune regulatory molecules, increased cytolytic activity, higher interferon-γ signature, and more abundant infiltration of immune cells. Cluster 1 and other cluster(s) in lung adenocarcinoma and lung squamous cell carcinoma had immunologically-hot and immunologically-cold tumor-immune microenvironments, respectively. Immunologically-hot cluster 1 subtype is hereafter referred to as "good-tumor-immune microenvironments" and the other subtypes as "bad-tumor-immune microenvironments." The "good-tumor-immune microenvironments" subtype in lung adenocarcinoma included a high fraction of CD8 T cells and memory B cells, but a low fraction of regulatory CD4 T cells and tumor-associated myeloid cells. Forward and backward application of our molecular subtyping to the Barcelona cohort revealed that transcriptome-based molecular subtyping is significantly associated with response to programmed cell death 1 blockade. CONCLUSIONS:Molecular stratification by transcriptome sequencing data in non-small cell lung cancer identifies distinct immunomolecular subtypes that predict the response to programmed cell death 1 blockade. 10.1016/j.jtcvs.2019.10.123