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Assessing APOBEC3 gene expression using immunohistochemical and bioinformatic approaches.

Immune cell deconvolution from molecular data. Relative proportions of immune cell subtypes are predicted from gene expression data using the Danaher method, and from methylation data using methylCIBERSORT. Differences between progressive and regressive groups are reported here.

APOBEC3 gene expression during senescence and cell cycle progression.

Supplementary Methods

Supplementary Table 1: Overview of data resource. Supplementary Table 2: List of antibodies used for immunofluorescence. Supplementary Table 3: List of antibodies used for western blotting. Supplementary Table 4. List of primers used for PCR.

Generation and characterization of A3A and A3B knockouts in TIIP cells.

A complete list of investigators in the Tracking Non-Small Cell Lung Cancer Evolution through Therapy (TRACERx) Consortium

Correlation between APOBEC3 expression and different measures of CIN.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non–small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution.Significance:This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution.This article is highlighted in the In This Issue feature, p. 2355

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

During human development, lungs develop their roles of gas exchange and barrier function. Recent single cell studies have focused on epithelial and mesenchymal cell types, but much less is known about the developing lung immune cells, although the airways are a major site of mucosal immunity after birth. An open question is whether tissue-resident immune cells play a role in shaping the tissue as it developsin utero. In order to address this, we profiled lung immune cells using scRNAseq, smFISH and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including ILCs, NK, myeloid cells and lineage progenitors. By the canalicular stage, we detected naive T lymphocytes high in cytotoxicity genes, and mature B lymphocytes, including B1 cells. Our analysis suggests that fetal lungs provide a niche for full B cell maturation. Given the abundance of immune cells, we investigated their possible effect on epithelial maturation and found that IL-1β drives epithelial progenitor exit from self-renewal and differentiation to basal cellsin vitro.In vivo, IL-1β-producing myeloid cells were found adjacent to epithelial tips, suggesting that immune cells may direct the developing lung epithelium.

Targeting early-stage lung cancer is vital to improve survival. However, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report, we study the role of Toll-like receptor 2 (TLR2), a regulator of oncogene-induced senescence, which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models, we show that TLR2 is active early in lung tumorigenesis, where it correlates with improved survival and clinical regression. Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses, such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors after Tlr2 loss. Last, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target., F.R.M. is funded by a Wellcome Trust clinical research fellowship through the Edinburgh Clinical Academic Track (ECAT) program (203913/Z/16/Z), a Wellcome Trust-ISSF3 award (IS3-R1.07 20/21), and a Wellcome Trust iTPA award (209710/Z/17/Z). J.C.A. core lab funding was received from Cancer Research UK (C47559/A16243, Training and Career Development Board – Career Development Fellowship), the University of Edinburgh (Chancellor’s Fellowship), and the Ministry of Science and Innovation of the Government of Spain (Proyecto PID2020-117860GB-100 financiado por MCIN/AEI/10.13039/501100011033). S.W. is supported by a Cancer Research UK senior fellowship (A29576). J.C. is supported by a Wellcome Trust clinical lectureship through the ECAT program (203913/Z/16/Z). M.M. is supported by a CRUK Edinburgh Centre Award (C157/A25140). S.V. and J.F.P. are funded by National Institute on Aging (NIA) grants (R01AG 68048-1 and UG3CA 268103-1).

Tobacco smoking causes lung cancer1–3, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the genome of lung cancer cells are well-documented6–10, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4–14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0–6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis. Whole-genome sequencing of normal bronchial epithelium from 16 individuals shows that tobacco smoking increases genomic heterogeneity, mutational burden and driver mutations, whereas stopping smoking promotes replenishment of the epithelium with near-normal cells.

BackgroundLung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival.Methods and resultsUsing an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-function leads to abnormally high cell proliferation during the earliest stages of pre-invasive disease and to the formation of significantly larger invasive tumours, suggesting accelerated disease progression.ConclusionTogether, our findings identify LRIG1 as a marker of basal airway progenitor cells with high proliferative potential and as a regulator of pre-invasive lung cancer progression. This work highlights the clinical relevance of LRIG1 and the potential of the NTCU-induced LUSC model for functional assessment of candidate tumour suppressors and oncogenes.

Targeting early-stage lung cancer is vital to improve overall survival. We previously identified Toll-like receptor 2 (TLR2) as a regulator of oncogene-induced senescence (OIS) and the senescence-associated secretory phenotype (SASP), both key for tumor suppression. Here, we demonstrate that TLR2 is widely expressed in human lung tumor epithelium where it correlates with improved survival and clinical regression. Using genetically engineered mouse models of lung cancer we have shown that Tlr2 is a tumor suppressor in lung cancer initiation via regulation of proliferation and the SASP. The SASP is integral in the regulation of immune surveillance of premalignant cells, and we observe impaired myeloid derived immune surveillance following Tlr2 loss. Lastly, we show that administration of a synthetic Tlr2 agonist significantly reduces preinvasive lung tumor growth. Our data highlight an unexpected tumor surveillance pathway in early-stage lung cancer with therapeutic potential.Statement of significanceLung cancer is a major cancer of unmet need. This study identifies a novel tumor suppressor mechanism in lung cancer. Not only does this highlight a potential therapeutic target for early-stage disease but also multiple secreted candidate biomarkers that could be exploited to augment lung cancer screening approaches.

It is well established that tobacco smoking causes lung cancer and this is thought to be the result of a progressive accumulation of genomic mutations induced by tobacco smoke exposure; however, until recently a detailed understanding of the effects of smoke exposure on the genomes of cells in the normal bronchial epithelium was lacking. To address this, we defined the genomic architecture of normal airway basal cells in children and non-smoking, smoking and ex-smoking adults. We discovered two populations of basal cells in individuals with a smoking history: those with high mutational burden, driver gene mutations and tobacco-associated mutational signatures, and those with low mutational burden, similar to basal cells from never smokers and with an absence of mutational signatures associated with tobacco exposure. These cells expand preferentially in ex-smokers, accounting for 20-40% of basal cells compared with 2-5% in current smokers. We hypothesise that these cells are cancer-protective and their expansion reduces the risk of lung cancer development after smoking cessation. Understanding the functional differences between high- and low-mutant airway basal cells and the mechanisms by which some cells resist the accumulation of smoking-induced mutations will be crucial to understanding the earliest stages of lung carcinogenesis. To address this, we performed RNAseq on a subset of whole-genome sequenced airway basal cells. Preliminary data show that expression of markers of stemness is significantly different between high- and low-mutant basal cells. In addition, initial analysis shows that pathways such as carcinogen metabolism and MHC class I antigen presentation are higher in high-mutant basal cells. Further analysis in more patients is ongoing. To complement these data, we expanded whole-genome sequenced basal cell clones in culture and performed a range of assays to assess their progenitor and differentiation capacity. We assessed proliferation, colony-forming efficiency, longevity and differentiation and found no apparent differences between low- and high-mutant basal cells. Initial analysis suggests that carcinogen metabolism and MHC class I antigen presentation may be key pathways in establishing heterogeneity in mutational burden and clonal dynamics in the normal airway epithelium. Additional assays and analysis are ongoing and will be the focus of future research. Citation Format: Kate H. Gowers, Sarah E. Clarke, Ayu Hutami Syaraf, Kenichi Yoshida, Moritz J. Przybilla, Hugh Selway, Adam Pennycuick, Peter J. Campbell, Sam M. Janes. Defining the mechanisms that lead to mutational heterogeneity in the normal respiratory epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3158.

Malignant pleural mesothelioma (MPM) is a rare, aggressive, and incurable cancer arising from the mesothelial lining of the pleura, with few available treatment options. We recently reported that loss of function of the nuclear deubiquitinase BRCA1-associated protein 1 (BAP1), a frequent event in MPM, is associated with sensitivity to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–mediated apoptosis. As a potential underlying mechanism, here we report that BAP1 negatively regulates the expression of TRAIL receptors: death receptor 4 (DR4) and death receptor 5 (DR5). Using tissue microarrays of tumor samples from MPM patients, we found a strong inverse correlation between BAP1 and TRAIL receptor expression. BAP1 knockdown increased DR4 and DR5 expression, whereas overexpression of BAP1 had the opposite effect. Reporter assays confirmed wt-BAP1, but not catalytically inactive BAP1 mutant, reduced promoter activities of DR4 and DR5, suggesting deubiquitinase activity is required for the regulation of gene expression. Co-immunoprecipitation studies demonstrated direct binding of BAP1 to the transcription factor Ying Yang 1 (YY1), and chromatin immunoprecipitation assays revealed BAP1 and YY1 to be enriched in the promoter regions of DR4 and DR5. Knockdown of YY1 also increased DR4 and DR5 expression and sensitivity to TRAIL. These results suggest that BAP1 and YY1 cooperatively repress transcription of TRAIL receptors. Our finding that BAP1 directly regulates the extrinsic apoptotic pathway will provide new insights into the role of BAP1 in the development of MPM and other cancers with frequent BAP1 mutations.

Background Lung cancer is the leading cause of cancer related deaths worldwide. The value of targeting early stage disease has been widely recognised to improve survival. Oncogene-induced senescence (OIS) is a stress response instigated following the activation of oncogenes and is a well-known tumour suppressor mechanism. OIS is abundant in pre-invasive lesions in murine lung cancer models, however is lost during the progression to malignancy. We previously identified a regulatory role for Toll-like receptor 2 (TLR2) in OIS and expression of the senescence-associated secretory phenotype (SASP), however the functional relevance of this has yet to be established. Methods We used genetically engineered mouse (GEM) models of lung cancer (KrasLSL-G12D/+ and KrasLSL-G12D/+;TP53fl/fl) on both wild-type and Tlr2 null backgrounds. Lung specific activation of mutant KrasG12D and TP53 loss was achieved upon intranasal infection with Cre-recombinase expressing adenovirus. Tumour burden, senescence markers and SASP expression were assessed by immunohistochemistry. Immune cell recruitment was measured using flow cytometry on whole lung single cell suspensions from tumour bearing mice. The expression of Tlr2 and associated SASP components were measured in human pre-invasive lung cancer samples that either progressed to invasive malignancy or regressed to normal epithelium. Results Tlr2 loss was associated with an increased tumour burden and reduced survival in our GEM model. Furthermore, Tlr2 loss caused significantly reduced epithelial expression of key senescence markers and SASP factors. However, immune cell recruitment was not affected suggesting this effect was cell intrinsic. Inhalational administration of a synthetic TLR2 agonist (Pam2CSK4) significantly reduced tumour burden in our GEM model. Molecular profiling of bronchoscopic biopsies of human pre-invasive lung lesions revealed increased TLR2 and SASP expression in samples that did not progress to invasive malignancy, suggesting a tumour suppressor role for TLR2-SASP signalling in human lung cancer. Conclusions We have identified TLR2 as a potential tumour suppressor in lung cancer via regulation of the SASP. Furthermore, we have highlighted a novel therapeutic strategy for the treatment of early stage lung cancer. SASP factors are released into the bloodstream and are ideal candidate biomarkers of pre-invasive disease and thus could potentially aid in stratifying lung cancer screening populations.

Malignant pleural mesothelioma (MPM) is a rare, aggressive, and incurable cancer arising from the mesothelial lining of the lungs with few treatment options. We recently reported loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1), a frequent event in MPM, is associated with sensitivity to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). As a potential underlying mechanism, here we report that BAP1 negatively regulates the expression of TRAIL receptors: death receptors 4 (DR4) and 5 (DR5). Using tissue microarray (TMAs) of tumour samples from MPM patients, we found a strong inverse correlation between BAP1 and TRAIL receptors.BAP1knockdown increased DR4 and DR5 expression, whereas overexpression of BAP1 had the opposite effect. Reporter assays confirmed wild-typeBAP1, but not catalytically-inactive mutantBAP1, reduced promoter activities ofDR4andDR5, suggesting deubiquinase activity plays an important role in the regulation of gene expression. Co-IP studies demonstrated direct binding of BAP1 and the transcription factor Ying Yang 1 (YY1) and ChIP assays revealed BAP1 and YY1 to be enriched in the promoter regions ofDR4andDR5. Notably, shRNA knockdown ofYY1also increased DR4 and DR5 expression, and sensitivity to TRAIL. These results demonstrate that BAP1 and YY1 together negatively regulate transcriptional activity of TRAIL receptors. BAP1 and YY1 may both therefore be strong therapeutic targets to enhance the efficacy of TRAIL-induced apoptosis.Statement of significanceWe describe how the most-frequently mutated tumour suppressor gene in mesothelioma regulates the response to TNF-related apoptosis-inducing ligand (TRAIL). These findings will accelerate a biomarker-driven cancer therapy.

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

Summary Tobacco smoking causes lung cancer1–3, driven by the 60+ carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the lung cancer genome have been well documented6–10, but we lack equivalent data for normal bronchial cells. We sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutation burden, adding 1000-10,000+ mutations/cell, massively increasing both within-subject and between-subject variance, and generating several distinct signatures of substitutions and indels. A population of cells in subjects with smoking history had mutation burdens equivalent to that expected for never-smokers: these cells had less damage from tobacco-specific mutational processes, were four-fold more frequent in ex-smokers than current smokers, and had significantly longer telomeres than their more mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged never-smokers. In current smokers, ≥25% of cells carried driver mutations and 0-6% cells had 2 or even 3 drivers. Thus, tobacco smoking increases mutation burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non–small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. Significance: This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution. This article is highlighted in the In This Issue feature, p. 2355

Before squamous cell lung cancer develops, pre-cancerous 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. While recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of pre-cancerous 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 harbour 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, TGF-beta signalling is overactive, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the CIS lesions as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. This study identifies mechanisms by which pre-cancerous 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.

Tobacco smoking causes lung cancer

Background Lung cancer is the leading cause of cancer death, of which 80-90% are attributed to tobacco smoking. Our understanding of how tobacco exposure affects mutational burden, mutational signatures, driver mutations and clonal dynamics in normal lung tissue is limited. Also, genetic differences between normal cells and cancer cells in lung has not been fully elucidated. Methods To access the landscape of somatic mutations in normal bronchial epithelia, we sequenced whole genomes of 632 single-cell-derived colonies of lung basal cells from 16 patients, including 3 children, 4 never-smokers, 6 ex-smokers and 3 current smokers. Five patients had squamous cell carcinomas or carcinoma in situ, which we also sequenced to compare the genetic alteration between normal and cancer cells. Results A positive correlation between the number of base substitutions and age was observed, and 22 somatic mutations accumulated per year (95% confidence interval:20-25; P=10−8). Previous or current smoking significantly increased mutational burden: 2330 substitutions in ex-smokers and 5300 in current smokers. In addition, tobacco smoking is massively increasing both between-subject and within-subject variance of mutation burden. A population of cells in subjects with smoking history had mutation burdens equivalent to that expected for never-smokers: these cells had less damage from tobacco-specific mutational processes, and were four-fold more frequent in ex-smokers than current smokers. Signature analysis revealed that the same mutational signatures seen in lung cancers operate both in patients with and without smoking history: endogeneous signatures, including COSMIC signatures 1 and 5, and APOBEC-related signatures. Three mutational signatures were largely restricted to current or ex-smokers, including known smoking-related COSMIC signature 4 characterized by C>A transversions, signature 16 characterized by T>C mutations with extremely strong transcription strand bias and a new signature characterized by T>A and T>C mutations. dN/dS method identified driver genes in normal bronchial epithelium, including NOTCH1, TP53 and FAT1, which were overlapped with those seen in squamous cell lung cancers and other normal squamous tissues such as esophagus and skin. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged never-smokers. In current smokers, ≥25% of cells carried driver mutations and 0-6% cells had 2 or even 3 drivers. Layering driver mutations onto phylogenetic trees revealed that driver mutations occurred in early life. Compared to the normal bronchial epithelial cells, lung cancers and precancerous lesions were characterized by extensive copy number changes or structural variants and distinct set of driver mutations. Conclusions Tobacco smoking increases mutation burden, cell-to-cell heterogeneity and driver mutations. Our data of genetic lesions in normal bronchial cells provides insights into genetic alterations that drive carcinogenesis in lung. Citation Format: Kenichi Yoshida, Kate HC Gowers, Henry Lee-Six, Deepak P. Chandrasekharan, Tim Coorens, Elizabeth F. Maughan, Kathryn Beal, Andrew Menzies, Fraser R. Millar, Elizabeth Anderson, Sarah E. Clarke, Adam Pennycuick, Ricky M. Thackeray, Colin R. Butler, Nobuyuki Kakiuchi, Tomonori Hirano, Robert E. Hynds, Michael R. Stratton, Inigo Martincorena, Sam M. Janes, Peter J. Campbell. Tobacco exposure and somatic mutations in normal bronchial epithelia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2342.

The molecular alterations that occur in cells before cancer is manifest are largely uncharted. Lung carcinoma in situ (CIS) lesions are the pre-invasive precursor to squamous cell carcinoma. Although microscopically identical, their future is in equipoise, with half progressing to invasive cancer and half regressing or remaining static. The cellular basis of this clinical observation is unknown. Here, we profile the genomic, transcriptomic, and epigenomic landscape of CIS in a unique patient cohort with longitudinally monitored pre-invasive disease. Predictive modeling identifies which lesions will progress with remarkable accuracy. We identify progression-specific methylation changes on a background of widespread heterogeneity, alongside a strong chromosomal instability signature. We observed mutations and copy number changes characteristic of cancer and chart their emergence, offering a window into early carcinogenesis. We anticipate that this new understanding of cancer precursor biology will improve early detection, reduce overtreatment, and foster preventative therapies targeting early clonal events in lung cancer. Postprint

Squamous cell carcinoma of the lung arises from preinvasive progenitors in the central airways. The archetypal model appears to be a stepwise morphological progression until there is invasion of the basement membrane. However, not every lesion appears to follow this course and many individuals can have stable disease, or indeed regress to normal epithelium. From our increased understanding of the molecular pathology it is becoming apparent that the respiratory epithelium accumulates progressive genetic and epigenetic insults in response to carcinogens. Still, little is known about how to predict those 'at risk' of progression, and it is likely that in the future molecular signatures will underpin prediction models of developing invasive lung cancer. Currently, autofluorescence bronchoscopy gives us the ability to follow the natural history of these lesions, with the prospect that detecting and treating lesions early may improve survival. However, treatment remains controversial, and radical therapies are offered to individuals with carcinoma in situ who may never develop invasive cancer. This has paved the way for the use of minimally invasive bronchoscopic treatments, which, while apparently effective, have not been tested in randomised controlled trials. In this paper we describe the known biology and natural history of preinvasive lesions and review the current treatment strategies.

Acute cellular rejection occurs frequently during the first few weeks following liver transplantation. During this period, its molecular phenotype is confounded by peri- and postoperative proinflammatory events. To unambiguously define the molecular profile associated with rejection, we collected sequential biological specimens from 55 patients at least 3 years after liver transplantation who developed rejection during trials of intentional immunosuppression withdrawal. We analyzed liver tissue and blood samples obtained before initiation of drug withdrawal and at rejection, alongside blood samples collected during the weaning process. Gene expression profiling was conducted using whole-genome microarrays and real-time polymerase chain reaction. Rejection resulted in distinct blood and liver tissue transcriptional changes in patients who were either positive or negative for hepatitis C virus (HCV). Gene expression changes were mostly independent from pharmacological immunosuppression, and their magnitude correlated with severity of histological damage. Differential expression of a subset of genes overlapped across all conditions. These were used to define a blood predictive model that accurately identified rejection in HCV-negative, but not HCV-positive, patients. Changes were detectable 1-2 mo before rejection was diagnosed. Our results provide insight into the molecular processes underlying acute cellular rejection in liver transplantation and help clarify the potential utility and limitations of transcriptional biomarkers in this setting.

Summary Aims: Epidermal growth factor receptor (EGFR) antagonists are particularly active in non-small cell lung cancer (NSCLC) patients with tumours bearing mutations in the EFGR gene. EGFR mutation prevalence is very low in squamous histology. Response rates using these drugs in patients with KRAS mutations are low, so available KRAS mutation information may aid treatment selection in the second-line setting. Since 2009, patients presenting to this hospital with non-squamous histology have been routinely screened for mutations in both the EGFR and KRAS genes, with results used to inform treatment. We present an analysis of 215 consecutive patients for whom EGFR mutation analysis was informative. Methodology: EGFR and KRAS mutations were identified using a COLD-PCR technique confirmed with sequencing, which makes no prior assumption about location of specific mutations. Results were correlated with clinical and demographic data from hospital records, where available. Results: The prevalence of patients with EGFR mutations was 14% and for KRAS mutations it was 27%. Despite the conventional understanding that EGFR and KRAS mutations are mutually exclusive, we identified two dual mutations. Of 29 patients identified with mutated EGFR, there were 3/8/8/10 mutations in exons 18/19/20/21 respectively. Exon 20 mutations were identified in a proportion exceeding many other series because of the unbiased mutation analysis used, and clinical benefit was seen in some of these. Of 23 different EGFR mutations identified, 11 have not previously been described in the literature. Conclusions: The high prevalence of EGFR, KRAS or both mutations (40%) in this non-squamous population tested in clinical practice supports a policy of routine screening for these mutations in NSCLC.

Introduction: The national Venous Thromboembolism (VTE) Prevention Programme in England was launched in 2010 and incorporates standardised guidance on risk assessment (RA) and thromboprophylaxis (TP) with a requirement for root cause analysis of all episodes of hospital associated thrombosis (HAT), defined as any VTE occurring whilst an inpatient or within 90 days of discharge. We previously reported findings of root cause analysis for HAT over 2010 - 2012, demonstrating that achieving a 90% risk assessment rate resulted in a significant reduction in the incidence of HAT. We update our findings on the impact of implementation of the national programme on the incidence of HAT, proportion of potentially preventable HAT episodes, and mortality from hospital-associated pulmonary embolism (PE). As appropriate TP only reduces the risk of VTE by two-thirds, we also looked at risk factors for TP failure. Methods: We examined HAT data collected from the root cause analysis programme at King's College Hospital from April 2011 to March 2015. Further data were gathered through retrospective review of patient notes. VTE risk factors for HAT attributed to TP failure were compared to a "non-HAT" group, (patients who received appropriate TP and did not develop HAT) drawn from VTE prevention audit data from 2013-2014. Episodes of HAT that developed following inadequate prescription or administration of either anticoagulant or mechanical TP were deemed as "potentially preventable" episodes. Results: Across the four-year study period there were 725 episodes of HAT, giving an incidence of 3.28 episodes per 1000 hospital admissions. There was no significant change in incidence from 2011-2015. The median age of the cohort was 64 years (IQR = 27 years). 56.7% (n = 411) of the HAT episodes were deep vein thromboses, of which 54.7% (n = 225) involved the proximal vasculature. PE accounted for 41.7% (n = 302) episodes, of which 10.9% (n = 33) were fatal events. HAT developed following medical, surgical or obstetric admission in 43.3% (n = 314), 54.6% (n = 396) and 2.1% (n = 15) respectively. VTE risk factors were present in 97.9% (n = 710) of patients with HAT with concomitant bleeding risk factors in 37.1% (n = 269). Consistently, the most common outcome of root cause analysis was TP failure (47.6% overall, n = 345) with no significant trend across the study period; 19.7% (n = 143) of episodes were attributed to inadequate anticoagulant TP, 26.1% (n = 189) to contraindication to anticoagulant TP, 4.4% (n = 32) to contraindication to all forms of TP, and 2.2% (n = 16) episodes were unexpected (HAT occurring in a patient without identifiable VTE risk factors). There has been a significant reduction in the proportion of potentially preventable HAT episodes from 38.2% (n = 66) in 2011-2012 to 20.3% (n = 39) in 2014-2015 (p < 0.001). Furthermore, the proportion of fatal PE reduced over the study period from 16.0% (n = 12) of HAT in 2011-2012 to 6.3% (n = 5) of HAT in 2014-2015 (p = 0.049). The audit of VTE prevention practice over 2013/14 included 515 patients, of which 423 (82.1%) received appropriate TP and did not develop HAT. Compared to this group, patients with HAT attributed to TP failure had more risk factors (3.1 vs. 2.7, p < 0.002), were more likely to be over 60 years of age (59.4% vs. 42.3%, p = 0.01), or to have had orthopaedic surgery(6.7% vs. 1.8%, p = 0.001). Discussion: Implementation of a comprehensive VTE prevention programme incorporating root cause analysis of HAT has led to a significant fall in the proportion of HAT that were potentially preventable with a corresponding reduction in mortality attributed to PE. However, there has been no change in the overall incidence of HAT with a rise in cases associated with TP failure. Further research is required to optimise TP in high VTE risk groups. Disclosures Arya: Bayer plc: Research Funding.