RESUMEN
Tumor microenvironments (TMEs) contain vast amounts of information on patient's cancer through their cellular composition and the spatial distribution of tumor cells and immune cell populations. Exploring variations in TMEs between patient groups, as well as determining the extent to which this information can predict outcomes such as patient survival or treatment success with emerging immunotherapies, is of great interest. Moreover, in the face of a large number of cell interactions to consider, we often wish to identify specific interactions that are useful in making such predictions. We present an approach to achieve these goals based on summarizing spatial relationships in the TME using spatial K functions, and then applying functional data analysis and random forest models to both predict outcomes of interest and identify important spatial relationships. This approach is shown to be effective in simulation experiments at both identifying important spatial interactions while also controlling the false discovery rate. We further used the proposed approach to interrogate two real data sets of Multiplexed Ion Beam Images of TMEs in triple negative breast cancer and lung cancer patients. The methods proposed are publicly available in a companion R package funkycells.
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Comunicación Celular , Microambiente Tumoral , Microambiente Tumoral/fisiología , Humanos , Comunicación Celular/fisiología , Biología Computacional/métodos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Algoritmos , Simulación por Computador , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias/inmunología , Neoplasias/patología , Modelos Biológicos , Femenino , Bosques AleatoriosRESUMEN
Spatial molecular data has transformed the study of disease microenvironments, though, larger datasets pose an analytics challenge prompting the direct adoption of single-cell RNA-sequencing tools including normalization methods. Here, we demonstrate that library size is associated with tissue structure and that normalizing these effects out using commonly applied scRNA-seq normalization methods will negatively affect spatial domain identification. Spatial data should not be specifically corrected for library size prior to analysis, and algorithms designed for scRNA-seq data should be adopted with caution.
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Perfilación de la Expresión Génica , Análisis de la Célula Individual , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Perfilación de la Expresión Génica/métodos , Algoritmos , BiologíaRESUMEN
We have previously identified alveolar type II cell as the cell-of-origin of KrasG12D-induced lung adenocarcinoma using cell lineage-specific inducible Cre mouse models. Using gain-of-function and loss-of-function genetic models, we discovered that active Notch signaling and low Sox2 levels dictate the ability of type II cells to proliferate and progress into lung adenocarcinoma upon KrasG12D activation. Here, we examine the phenotype of type II cells after Kras activation and find evidence for proliferation of cells that coexpress type I and type II markers. Three-dimensional organoid culture and transplantation studies determine that these dual-positive cells are highly plastic and tumor initiating in vivo. RNA sequencing analysis reveals that these dual-positive cells are enriched in Ras/MAPK, EGFR, and Notch pathways. Furthermore, the proliferation of these cells requires active Notch signaling and is inhibited by genetic/chemical Sox2 upregulation. Our findings could provide new therapeutic strategies to target KRAS-activated lung adenocarcinomas. SIGNIFICANCE: Identification of progenitor like tumor-initiating cells in KRAS-mutant lung adenocarcinoma may allow development of novel targeted therapeutics.
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Adenocarcinoma del Pulmón , Adenocarcinoma , Neoplasias Pulmonares , Ratones , Animales , Proteínas Proto-Oncogénicas p21(ras)/genética , Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Plasticidad de la Célula , Proliferación Celular/genética , Adenocarcinoma del Pulmón/genéticaRESUMEN
The discovery of frequent 8p11-p12 amplifications in squamous cell lung cancer (SQLC) has fueled hopes that FGFR1, located inside this amplicon, might be a therapeutic target. In a clinical trial, only 11% of patients with 8p11 amplification (detected by FISH) responded to FGFR kinase inhibitor treatment. To understand the mechanism of FGFR1 dependency, we performed deep genomic characterization of 52 SQLCs with 8p11-p12 amplification, including 10 tumors obtained from patients who had been treated with FGFR inhibitors. We discovered somatically altered variants of FGFR1 with deletion of exons 1-8 that resulted from intragenic tail-to-tail rearrangements. These ectodomain-deficient FGFR1 variants (ΔEC-FGFR1) were expressed in the affected tumors and were tumorigenic in both in vitro and in vivo models of lung cancer. Mechanistically, breakage-fusion-bridges were the source of 8p11-p12 amplification, resulting from frequent head-to-head and tail-to-tail rearrangements. Generally, tail-to-tail rearrangements within or in close proximity upstream of FGFR1 were associated with FGFR1 dependency. Thus, the genomic events shaping the architecture of the 8p11-p12 amplicon provide a mechanistic explanation for the emergence of FGFR1-driven SQLC. Specifically, we believe that FGFR1 ectodomain-deficient and FGFR1-centered amplifications caused by tail-to-tail rearrangements are a novel somatic genomic event that might be predictive of therapeutically relevant FGFR1 dependency.
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Carcinoma de Pulmón de Células no Pequeñas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Amplificación de Genes , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Células Epiteliales/metabolismoRESUMEN
In August 2021, the SOLEIL storage ring was restarted after the summer shutdown with a new bending magnet made entirely of permanent magnets. Producing a magnetic field of 2.8â T, it replaced one of the 32 electromagnetic dipoles (magnetic field of 1.7â T) of the ring to allow the ROCK beamline to exploit more intense photon fluxes in the hard X-ray range, thus improving the time resolution performances of the beamline for experiments carried out above 20â keV. The reduction of the new dipole magnetic gap required to produce the higher field has led to the construction and installation of a new vacuum vessel. The realization of the new dipole with permanent magnets was a technological feat due to the very strong magnetic forces. The permanent-magnet assembly required dedicated tools to be designed and constructed. Thanks to accurate magnetic measurements, a precise modelization of the new dipole was performed to identify its effects on the electron beam dynamics. The first measurements carried out on the ROCK beamline have highlighted the expected increase in photon flux, and the operation performances remain unchanged for the other beamlines. Here, the major developments and results of this innovative project are described in terms of technology, electron beam dynamics and photon beam performance on the ROCK beamline.
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Imanes , Sincrotrones , Rayos X , FotonesRESUMEN
Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer death worldwide. Immunotherapy with immune checkpoint inhibitors (ICI) has significantly improved outcomes in some patients, however 80-85% of patients receiving immunotherapy develop primary resistance, manifesting as a lack of response to therapy. Of those that do have an initial response, disease progression may occur due to acquired resistance. The make-up of the tumour microenvironment (TME) and the interaction between tumour infiltrating immune cells and cancer cells can have a large impact on the response to immunotherapy. Robust assessment of the TME with accurate and reproducible methods is vital to understanding mechanisms of immunotherapy resistance. In this paper we will review the evidence of several methodologies to assess the TME, including multiplex immunohistochemistry, imaging mass cytometry, flow cytometry, mass cytometry and RNA sequencing.
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Tissue-resident memory T (TRM) cells provide immune defense against local infection and can inhibit cancer progression. However, it is unclear to what extent chronic inflammation impacts TRM activation and whether TRM cells existing in tissues before tumor onset influence cancer evolution in humans. We performed deep profiling of healthy lungs and lung cancers in never-smokers (NSs) and ever-smokers (ESs), finding evidence of enhanced immunosurveillance by cells with a TRM-like phenotype in ES lungs. In preclinical models, tumor-specific or bystander TRM-like cells present prior to tumor onset boosted immune cell recruitment, causing tumor immune evasion through loss of MHC class I protein expression and resistance to immune checkpoint inhibitors. In humans, only tumors arising in ES patients underwent clonal immune evasion, unrelated to tobacco-associated mutagenic signatures or oncogenic drivers. These data demonstrate that enhanced TRM-like activity prior to tumor development shapes the evolution of tumor immunogenicity and can impact immunotherapy outcomes.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Células T de Memoria , Memoria Inmunológica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Pulmón , Linfocitos T CD8-positivosRESUMEN
Introduction: Tumour mutational burden (TMB) is an important emerging biomarker for immune checkpoint inhibitors (ICI). The stability of TMB values across distinct EBUS tumour regions is not well defined in advanced lung cancer patients. Methods: This study included a whole-genome sequencing cohort (n=11, LxG cohort) and a targeted Oncomine TML panel cohort (n=10, SxD cohort), where paired primary and metastatic samples were obtained by endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA). Results: The LxG cohort displayed a strong correlation between the paired primary and metastatic sites, with a median TMB score of 7.70 ± 5.39 and 8.31 ± 5.88 respectively. Evaluation of the SxD cohort demonstrated greater inter-tumoural TMB heterogeneity, where Spearman correlation between the primary and metastatic sites fell short of significance. Whilst median TMB scores were not significantly different between the two sites, 3 out of 10 paired samples were discordant when using a TMB cut-off of 10 mutations per Mb. In addition, PD-L1 copy number and KRAS mutations were assessed, demonstrating the feasibility of performing multiple molecular tests relevant to ICI treatment using a single EBUS sample. We also observed good consistency in PD-L1 copy number and KRAS mutation, where cut-off estimates were consistent across the primary and metastatic sites. Conclusions: Assessment of TMB acquired by EBUS from multiple sites is highly feasible and has the potential to improve accuracy of TMB panels as a companion diagnostic test. We demonstrate similar TMB values across primary and metastatic sites, however 3 out of 10 samples displayed inter-tumoural heterogeneity that would alter clinical management.
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Evading immune destruction is one of the hallmarks of cancer. A key mechanism of immune evasion deployed by tumour cells is to reduce neoantigen presentation through down-regulation of the antigen presentation machinery. MHC-I and MHC-II proteins are key components of the antigen presentation machinery responsible for neoantigen presentation to CD8+ and CD4+ T lymphocytes, respectively. Their expression in tumour cells is modulated by a complex interplay of genomic, transcriptomic and post translational factors involving multiple intracellular antigen processing pathways. Ongoing research investigates mechanisms invoked by cancer cells to abrogate MHC-I expression and attenuate anti-tumour CD8+ cytotoxic T cell response. The discovery of MHC-II on tumour cells has been less characterized. However, this finding has triggered further interest in utilising tumour-specific MHC-II to harness sustained anti-tumour immunity through the activation of CD4+ T helper cells. Tumour-specific expression of MHC-I and MHC-II has been associated with improved patient survival in most clinical studies. Thus, their reactivation represents an attractive way to unleash anti-tumour immunity. This review provides a comprehensive overview of physiologically conserved or novel mechanisms utilised by tumour cells to reduce MHC-I or MHC-II expression. It outlines current approaches employed at the preclinical and clinical trial interface towards reversing these processes in order to improve response to immunotherapy and survival outcomes for patients with cancer.
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Presentación de Antígeno , Neoplasias , Linfocitos T CD4-Positivos , Humanos , Vigilancia Inmunológica , InmunoterapiaRESUMEN
Understanding intratumoral heterogeneity-the molecular variation among cells within a tumor-promises to address outstanding questions in cancer biology and improve the diagnosis and treatment of specific cancer subtypes. Single-cell analyses, especially RNA sequencing and other genomics modalities, have been transformative in revealing novel biomarkers and molecular regulators associated with tumor growth, metastasis and drug resistance. However, these approaches fail to provide a complete picture of tumor biology, as information on cellular location within the tumor microenvironment is lost. New technologies leveraging multiplexed fluorescence, DNA, RNA and isotope labeling enable the detection of tens to thousands of cancer subclones or molecular biomarkers within their native spatial context. The expeditious growth in these techniques, along with methods for multiomics data integration, promises to yield a more comprehensive understanding of cell-to-cell variation within and between individual tumors. Here we provide the current state and future perspectives on the spatial technologies expected to drive the next generation of research and diagnostic and therapeutic strategies for cancer.
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Perfilación de la Expresión Génica/métodos , Espectrometría de Masas/métodos , Neoplasias/diagnóstico por imagen , Proteínas/análisis , Animales , Humanos , Ratones Transgénicos , Imagen Multimodal , Neoplasias/genética , Neoplasias/patología , Análisis de la Célula Individual/métodos , Microambiente TumoralRESUMEN
T cell memory is critical in controlling infection and plays an important role in anti-tumor responses in solid cancers. While effector memory and central memory T cells circulate and patrol non-lymphoid and lymphoid organs respectively, tissue resident memory T cells (TRM) permanently reside in tissues and provide local protective immune responses. In a number of solid tumors, tumor-specific T cell memory responses likely play an important role in keeping tumors in check, limiting cancer cell dissemination and reducing risk of relapse. In non-small cell lung cancer (NSCLC), a subset of tumor infiltrating lymphocytes (TILs) display phenotypic and functional characteristics associated with lung TRM (TRM-like TILs), including the expression of tissue-specific homing molecules and immune exhaustion markers. High infiltration of TRM-like TILs correlates with better survival outcomes for lung cancer patients, indicating that TRM-like TILs may contribute to anti-tumor responses. However, a number of TRM-like TILs do not display tumor specificity and the exact role of TRM-like TILs in mediating anti-tumor response in lung cancer is unclear. Here we review the characteristics of TRM-like TILs in lung cancer, the role these cells play in mediating anti-tumor immunity and the therapeutic implications of TRM-like TILs in the use and development of immunotherapy for lung cancer.
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Lung cancer is the leading cause of cancer death worldwide, with approximately 1.6 million cancer related deaths each year. Prognosis is best in patients with early stage disease, though even then five-year survival is only 55% in some groups. Median survival for advanced non-small cell lung cancer (NSCLC) is 8-12 months with conventional treatment. Immune checkpoint inhibitor (ICI) therapy has revolutionised the treatment of NSCLC with significant long-term improvements in survival demonstrated in some patients with advanced NSCLC. However, only a small proportion of patients respond to ICI, suggesting the need for further techniques to harness the potential of ICI therapy. Thermal ablation utilizes the extremes of temperature to cause tumour destruction. Commonly used modalities are radiofrequency ablation (RFA), cryoablation and microwave ablation (MWA). At present thermal ablation is reserved for curative-intent therapy in patients with localized NSCLC who are unable to undergo surgical resection or stereotactic ablative body radiotherapy (SABR). Limited evidence suggests that thermal ablative modalities can upregulate an anticancer immune response in NSCLC. It is postulated that thermal ablation can increase tumour antigen release, which would initiate and upregulated steps in the cancer immunity cycle required to elicit an anticancer immune response. This article will review the current thermal ablative techniques and their ability to modulate an anti-cancer immune response with a view of using thermal ablation in conjunction with ICI therapy.
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Apoptosis can potently defend against intracellular pathogens by directly killing microbes and eliminating their replicative niche. However, the reported ability of Mycobacterium tuberculosis to restrict apoptotic pathways in macrophages in vitro has led to apoptosis being dismissed as a host-protective process in tuberculosis despite a lack of in vivo evidence. Here we define crucial in vivo functions of the death receptor-mediated and BCL-2-regulated apoptosis pathways in mediating protection against tuberculosis by eliminating distinct populations of infected macrophages and neutrophils and priming T cell responses. We further show that apoptotic pathways can be targeted therapeutically with clinical-stage compounds that antagonize inhibitor of apoptosis (IAP) proteins to promote clearance of M. tuberculosis in mice. These findings reveal that any inhibition of apoptosis by M. tuberculosis is incomplete in vivo, advancing our understanding of host-protective responses to tuberculosis (TB) and revealing host pathways that may be targetable for treatment of disease.
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Apoptosis/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Neutrófilos/inmunología , Tuberculosis Pulmonar/inmunología , Animales , Caspasa 8/genética , Caspasa 8/metabolismo , Línea Celular , Dipéptidos/uso terapéutico , Humanos , Indoles/uso terapéutico , Activación de Linfocitos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/microbiología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Linfocitos T/inmunología , Tiazoles/uso terapéutico , Tuberculosis Pulmonar/tratamiento farmacológicoRESUMEN
Development of a branching tree in the embryonic lung is crucial for the formation of a fully mature functional lung at birth. Sox9+ cells present at the tip of the primary embryonic lung endoderm are multipotent cells responsible for branch formation and elongation. We performed a genetic screen in murine primary cells and identified aurora kinase b (Aurkb) as an essential regulator of Sox9+ cells ex vivo. In vivo conditional knockout studies confirmed that Aurkb was required for lung development but was not necessary for postnatal growth and the repair of the adult lung after injury. Deletion of Aurkb in embryonic Sox9+ cells led to the formation of a stunted lung that retained the expression of Sox2 in the proximal airways, as well as Sox9 in the distal tips. Although we found no change in cell polarity, we showed that loss of Aurkb or chemical inhibition of Aurkb caused Sox9+ cells to arrest at G2/M, likely responsible for the lack of branch bifurcation. This work demonstrates the power of genetic screens in identifying novel regulators of Sox9+ progenitor cells and lung branching morphogenesis.
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Aurora Quinasa B/genética , Aurora Quinasa B/metabolismo , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Pulmón/embriología , Factor de Transcripción SOX9/metabolismo , Animales , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Organogénesis , Factor de Transcripción SOX9/genéticaRESUMEN
Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.
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Antimitóticos/farmacología , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Animales , Antimitóticos/farmacocinética , Antimitóticos/toxicidad , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Células Hep G2 , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Mitosis/efectos de los fármacos , Neoplasias/patología , Células PC-3 , Ratas Sprague-Dawley , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27-CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. SIGNIFICANCE: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer.See related commentary by Krysan et al., p. 1442.This article is highlighted in the In This Issue feature, p. 1426.
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Carcinoma de Células Escamosas/inmunología , Vigilancia Inmunológica/inmunología , Neoplasias Pulmonares/inmunología , HumanosAsunto(s)
Adhesión Celular/fisiología , Metástasis de la Neoplasia/patología , Células Madre Neoplásicas/fisiología , Nicho de Células Madre/fisiología , Microambiente Tumoral/fisiología , Humanos , Neoplasias/patología , Células Madre Neoplásicas/patología , Neutrófilos/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiologíaRESUMEN
RNA-seq datasets can contain millions of intron reads per library that are typically removed from downstream analysis. Only reads overlapping annotated exons are considered to be informative since mature mRNA is assumed to be the major component sequenced, especially for poly(A) RNA libraries. In this study, we show that intron reads are informative, and through exploratory data analysis of read coverage that intron signal is representative of both pre-mRNAs and intron retention. We demonstrate how intron reads can be utilized in differential expression analysis using our index method where a unique set of differentially expressed genes can be detected using intron counts. In exploring read coverage, we also developed the superintronic software that quickly and robustly calculates user-defined summary statistics for exonic and intronic regions. Across multiple datasets, superintronic enabled us to identify several genes with distinctly retained introns that had similar coverage levels to that of neighbouring exons. The work and ideas presented in this paper is the first of its kind to consider multiple biological sources for intron reads through exploratory data analysis, minimizing bias in discovery and interpretation of results. Our findings open up possibilities for further methods development for intron reads and RNA-seq data in general.
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Neoplasias , Tejido Parenquimatoso , Células Madre , Humanos , Tejido Parenquimatoso/citologíaRESUMEN
Our understanding of genomic heterogeneity in lung cancer is largely based on the analysis of early-stage surgical specimens. Here we used endoscopic sampling of paired primary and intrathoracic metastatic tumors from 11 lung cancer patients to map genomic heterogeneity inoperable lung cancer with deep whole-genome sequencing. Intra-patient heterogeneity in driver or targetable mutations was predominantly in the form of copy number gain. Private mutation signatures, including patterns consistent with defects in homologous recombination, were highly variable both within and between patients. Irrespective of histotype, we observed a smaller than expected number of private mutations, suggesting that ancestral clones accumulated large mutation burdens immediately prior to metastasis. Single-region whole-genome sequencing of from 20 patients showed that tumors in ever-smokers with the strongest tobacco signatures were associated with germline variants in genes implicated in the repair of cigarette-induced DNA damage. Our results suggest that lung cancer precursors in ever-smokers accumulate large numbers of mutations prior to the formation of frank malignancy followed by rapid metastatic spread. In advanced lung cancer, germline variants in DNA repair genes may interact with the airway environment to influence the pattern of founder mutations, whereas similar interactions with the tumor microenvironment may play a role in the acquisition of mutations following metastasis.