Esophageal adenocarcinoma (EAC) has a poor outcome, and targeted therapy trials have thus far been disappointing due to a lack of robust stratification methods. Whole-genome sequencing (WGS) analysis of 129 cases demonstrates that this is a heterogeneous cancer dominated by copy number alterations with frequent large scale rearrangements. Co-amplification of receptor tyrosine kinases (RTKs) and/or downstream mitogenic activation is almost ubiquitous; thus tailored combination RTKi therapy might be required, as we demonstrate in vitro. However, mutational signatures reveal three distinct molecular subtypes with potential therapeutic relevance, which we verify in an independent cohort (n=87): i) enriched for BRCA signature with prevalent defects in the homologous recombination pathway; ii) dominant T>G mutational pattern associated with a high mutational load and neoantigen burden; iii) C>A/T mutational pattern with evidence of an ageing imprint. These subtypes could be ascertained using a clinically applicable sequencing strategy (low coverage) as a basis for therapy selection.

Esophageal adenocarcinoma (EAC) incidence is increasing while 5-year survival rates remain less than 15%. A lack of experimental models has hampered progress. We have generated clinically annotated EAC organoid cultures that recapitulate the morphology, genomic, and transcriptomic landscape of the primary tumor including point mutations, copy number alterations, and mutational signatures. Karyotyping of organoid cultures has confirmed polyclonality reflecting the clonal architecture of the primary tumor. Furthermore, subclones underwent clonal selection associated with driver gene status. Medium throughput drug sensitivity testing demonstrates the potential of targeting receptor tyrosine kinases and downstream mediators. EAC organoid cultures provide a pre-clinical tool for studies of clonal evolution and precision therapeutics.

Data availability. The WGS and RNA expression data can be found at the European Genome-phenome Archive (EGA) under accessions EGAD00001004417 and EGAD00001004423, respectively. Code availability. Code associated with the analysis is available upon request. Ethics. The study was registered (UKCRNID 8880), approved by the Institutional Ethics Committees (REC 07/H0305/52 and 10/ H0305/1), and all subjects gave individual informed consent. Reporting summary. Additional information is included in the Life Sciences Reporting Summary, which details exact software and biological materials used and efforts made to ensure reproducibility of results. Author contributions RCF and AMF conceived the overall study. AMF and SJ analyzed the genomic data and performed statistical analyses. RCF, AMF and XL designed the experiments. AMF, XL and JM performed the experiments. GC contributed to the structural variant analysis and data visualization. SK helped compile the clinical data and aided statistical analyses. JP and SA produced and QC'ed the RNA-seq data. EO aided the whole genome sequencing of EAC cell lines. SM and NG coordinated the clinical centres and were responsible for sample collections. ME benchmarked our mutation calling pipelines. MO led the pathological sample QC for sequencing. LB and GD constructed and managed the sequencing alignment and variant calling pipelines. RCF and ST supervised the research. RCF and ST obtained funding. AMF and RCF wrote the manuscript. All authors approved the manuscript.

14Esophageal Adenocarcinoma (EAC) is a poor prognosis cancer type with rapidly rising incidence. Our 15 understanding of genetic events which drive EAC development is limited and there are few molecular 16 biomarkers for prognostication or therapeutics. We have accumulated a cohort of 551 genomically 17 characterised EACs (73% WGS and 27% WES) with clinical annotation and matched RNA-seq. Using a 18 variety of driver gene detection methods we discover 65 EAC drivers (66% novel) and describe 19 mutation and CNV types with specific functional impact. We identify a mean of 3.7 driver events per 20 case derived almost equally from copy number events and mutations. We compare driver mutation 21 rates to the exome-wide mutational excess calculated using Non-synonymous vs Synonymous 22 mutation rates (dNdS). We see mutual exclusivity or co-occurrence of events within and between a 23 number of EAC pathways (GATA factors, Core Cell cycle genes, TP53 regulators and the SWI/SNF 24 complex) suggestive of important functional relationships. These driver variants correlate with tumour 25 differentiation, sex and prognosis. Poor prognostic indicators (SMAD4, GATA4) are verified in 26 independent cohorts with significant predictive value. Over 50% of EACs contain sensitising events for 27 CDK4/6 inhibitors which are highly correlated with clinically relevant sensitivity in a panel EAC cell 28 lines. 29 30 simplest of these features is the tendency of a mutation to co-occur with other mutations in the 48 same gene at a high frequency, as detected by MutsigCV 9 . MutsigCV has been applied on several 49 occasions to EAC cohorts 6,10,11 and has identified ten known cancer genes as high confidence EAC 50 drivers (TP53, CDKN2A, SMAD4, ARID1A, ERBB2, KRAS, PIK3CA, SMARCA4, CTNNB1 and FBXW7). 51 However these analyses leave most EAC cases with only one known driver mutation, usually TP53, 52 due to the low frequency at which other drivers occur. Equivalent analyses in other cancer types 53 have identified three or four drivers per case 12,13 . Similarly, detection of copy number driver events 54 in EAC has relied on identifying regions of the genome recurrently deleted or amplified, as detected 55 by GISTIC 10,14-17 . However, GISTIC identifies relatively large regions of the genome, containing 56 hundreds of genes, with little indication of which specific gene-copy number aberrations (CNAs) may 57 actually confer a selective advantage. There are also several non-selection based mechanisms which 58 can cause recurrent CNAs, such as fragile sites where a low density of DNA replication origins causes 59 frequent structural events at a particular loci. These have not been differentiated properly from 60 selection based recurrent CNAs 18 . 61Without proper annotation of the genomic variants which drive the biology of EAC tumours 62we are left with a very large number of events, most of which are likely to be inconsequential, 63 making it extremely difficult to detect statistical associations between genomic variants and various 64 biologi...

Oesophageal adenocarcinoma (OAC) is one of the ten most prevalent forms of cancer and is showing a rapid increase in incidence and yet exhibits poor survival rates. Compared to many other common cancers, the molecular changes that occur in this disease are relatively poorly understood. However, genes encoding chromatin remodeling enzymes are frequently mutated in OAC. This is consistent with the emerging concept that cancer cells exhibit reprogramming of their chromatin environment which leads to subsequent changes in their transcriptional profile. Here, we have used ATAC-seq to interrogate the chromatin changes that occur in OAC using both cell lines and patient-derived material. We demonstrate that there are substantial changes in the regulatory chromatin environment in the cancer cells and using this data we have uncovered an important role for ETS and AP1 transcription factors in driving the changes in gene expression found in OAC cells.

Background & Aims-Esophageal adenocarcinomas (EAC) are heterogeneous and often preceded by Barrett's esophagus (BE). Many genomic changes have been associated with development of BE and EAC, but little is known about epigenetic alterations. We performed *

Data AvailabilitySequencing data that support the findings of this paper have been deposited in the European Genome-phenome Archive with the accession code EGAD00001005434. Code AvailabilityAll code required to reproduce the analysis outline in this manuscript can be found in the main and supplementary methods. There are no restrictions to the accessibility of this code. Author ContributionsAN designed and implemented the rapid autopsy study, collected the samples, performed the experiments, analyzed data and wrote the manuscript. MG and S.D.P contributed expertise in pathology and sample collection for the rapid autopsy study. ID-B and NG assisted in study implementation, and along with JC, assisted with sample collection at autopsy. M.S performed the structural variant analysis. M.D.E performed genomic data generation and QC. LB conducted data management. XL, PL-S and JW were involved with autopsy sample collection, advice on experiments and data analysis, and XL contributed to experiments, paper writing, and figure design. LA and IM assisted with data analysis. NG assisted with study Implementation. SMac coordinated the sequencing of samples from the OCCAMS project and contributed to paper writing. SM and AM provided pathology data. TT, SG, LP and DG assisted in implementation and ethical conduct of the autopsy study. R.H.H and AH were involved in surgical sample collection and providing surgical expertise. M.R.S contributed to critical evaluation of the study data and manuscript. D.C.W was responsible for data analysis, paper writing, and assuring integrity of data. The OCCAMS consortium was the vehicle through which the infrastructure and funding was obtained to support the study and the consortium contributed to discussions on the ICGC data and the clinical ramifications. R.C.F provided grant funding and was responsible for study design, supervision of the project, writing the paper and assuring integrity of the data.

MYC oncoproteins deliver a potent oncogenic stimulus in several human cancers, making them major targets for drug development, but efforts to deliver clinically practical therapeutics have not yet been realized. In childhood cancer, aberrant expression of MYC and MYCN genes delineates a group of aggressive tumours responsible for a major proportion of pediatric cancer deaths. We designed a chemical-genetic screen that identifies compounds capable of enhancing proteasomal elimination of MYCN oncoprotein. We isolated several classes of compound that selectively kill MYCN expressing cells and we focus on inhibitors of PI3K/mTOR pathway in this study. We show that PI3K/mTOR inhibitors selectively killed MYCN-expressing neuroblastoma tumor cells, and induced significant apoptosis of transgenic MYCN-driven neuroblastoma tumors concomitant with elimination of MYCN protein in vivo. Mechanistically, the ability of these compounds to degrade MYCN requires complete blockade of mTOR but not PI3 kinase activity and we highlight NVP-BEZ235 as a PI3K/mTOR inhibitor with an ideal activity profile. These data establish that MYCN expression is a marker indicative of likely clinical sensitivity to mTOR inhibition, and provide a rationale for the selection of clinical candidate MYCN-destabilizers likely to be useful for the treatment of MYCN-driven cancers.