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Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer1–6. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett’s oesophagus. These data included 206 biopsies in Barrett’s oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case–control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett’s-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection.

Background Intra-tumour heterogeneity has been well-described in oesophageal adenocarcinoma and Barrett's oesophagus (BE) segments also consist of multiple clones (groups of cells which share mutations). In BE there is some suggestion that the number of individual genetic clones within the segment and the diversity of the segment can be predictors of progression to cancer. However, this was with pre- whole genome sequencing (WGS) methods. In this study we consider the detailed spatial maps of ten patients with different disease trajectories, using WGS, to determine the heterogeneity and correlate it with their progression. Methods We performed WGS (50x) on 35 high-quality, frozen, endoscopic biopsies taken spatially from ten cases (8 males, 2 females, median age 75 (range 31–83). This included 4 long-term non-dysplastic (ND) patients with long BE segments and 6 patients with dysplasia. We used the copy number and purity information (from Battenberg) and the somatic mutation clustering information (from DPClust) to infer the clonal and subclonal architecture of individual biopsies and looked at the spatial relationship between biopsies within the segment. Results In both ND and dysplastic BE we saw cells with unrelated patterns of mutations, or clones, implying completely separate origins for different parts of the segments. This suggests independent or parallel evolution. There was a higher burden of shared (truncal) mutations in the dysplastic biopsies (p=0.049), indicating a more recent clonal sweep with some clones being outcompeted. Dysplastic samples had more subclonal copy number aberrations and more clonal driver mutations. In addition, most of the driver gene alterations in the dysplastic trees occurred early, whereas CDKN2A, the p16 locus, was the only truncal mutation found on the ND clone trees. We did not find the number of clones or the diversity of the segment to differ between the two groups. Conclusions Each individual patient showed complex, distinct genomic features and evolutionary trajectories. Some cases arose from a single ancestral clone, while others had few shared mutational variants suggesting multiple clones. Importantly, in this sample of cases we did not see any trend that the clonal diversity or number of clones correlated with progression to cancer. This is different to previous studies and highlights the importance of multiple sampling to determine the propensity for cancer progression even if the samples look similar histologically.

BACKGROUNDOncogenes are commonly amplified on extrachromosomal DNA (ecDNA) contributing to poor outcomes for patients. Currently, the chronology of ecDNA development is not known. We studied the origination and evolution of ecDNA in patients with Barrett’s esophagus (BE) who progressed to esophageal adenocarcinoma (EAC).METHODSWe analyzed whole-genome sequencing (WGS) data from a BE surveillance cohort and EAC patients at Cambridge University UK (n=206 patients). We also analyzed WGS data from biopsies taken at two time points from multiple sites in the esophagus from 80 patients enrolled in a case-control study at the Fred Hutchinson Cancer Center (FHCC) - 40 BE patients who progressed to EAC and 40 who did not.RESULTSecDNA was detected in 24% and 43% of BE patients with BE-associated early and late-stage EAC, respectively, in the Cambridge cross-sectional cohort. ecDNA was found in 33% of all FHCC BE patients who developed cancer, either prior to, or at EAC diagnosis. ecDNA was strongly associated with patients who developed cancer, in contrast with FHCC BE patients who did not progress (odds ratio, 18.8, CI – 2.3-152, p=3.3×10-4). ecDNAs were enriched for oncogenes and immunomodulatory genes and could be detected early in the transition from high-grade dysplasia to cancer and increased in copy number and complexity over time.CONCLUSIONSecDNAs can develop before a diagnosis of cancer in BE patients and are strongly selected for during the evolution to EAC. ecDNAs promote diverse oncogene and immunomodulatory gene amplification during EAC development and progression.

Prognostication in oesophageal cancer on the basis of preoperative variables is challenging. Many of the accepted predictors of survival are only derived after surgical treatment and may be influenced by neoadjuvant therapy. This study aims to explore the relationship between pre-treatment endoscopic tumour morphology and postoperative survival.Patients with endoscopic descriptions of tumours were identified from the prospectively managed databases including the OCCAMS database. Tumours were classified as exophytic, ulcerating or stenosing. Kaplan Meier survival analysis and multivariable Cox regression analyses were performed to determine hazard ratios (HR) with 95% confidence intervals.262 patients with oesophageal adenocarcinoma undergoing potentially curative resection were pooled from St Thomas' Hospital (161) and the OCCAMS database (101). There were 70 ulcerating, 114 exophytic and 78 stenosing oesophageal adenocarcinomas. Initial tumour staging was similar across all groups (T3/4 tumours 71.4%, 70.2%, 74.4%). Median survival was 55 months, 51 months and 36 months respectively (p 0.001). Rates of lymphovascular invasion (P = 0.0176), pathological nodal status (P = 0.0195) and pathological T stage (P = 0.0007) increased from ulcerating to exophytic to stenosing lesions. Resection margin positivity was 21.4% in ulcerating tumours compared to 54% in stenosing tumours (p 0.001). When compared to stenosing lesions, exophytic and ulcerating lesions demonstrated a significant survival advantage on multivariable analysis (HR 0.56 95% CI 0.31-0.93, HR 0.42 95% CI 0.21-0.82).This study demonstrates that endoscopic morphology may be an important pre-treatment prognostic factor in oesophageal cancer. Ulcerating, exophytic and stenosing tumours may represent different pathological processes and tumour biology.

Oncogene amplification on extrachromosomal DNA (ecDNA) drives rapid tumor evolution, treatment resistance, and poor outcomes for patients. Computational tools can detect ecDNA in whole-genome sequencing (WGS) data from biopsies. However, the lack of longitudinal studies tracking patients from pre-cancer to cancer have made it difficult to determine how ecDNAs contribute to the malignant transformation. Two independent surveillance studies of Barrett’s esophagus (BE) patients enabled us to study this transition, as BE is the precursor lesion of esophageal adenocarcinoma (EAC). The studies included a longitudinal case-control study and an independent, cross-sectional surveillance cohort, both with histological correlatives, providing an unprecedented opportunity to study the role of ecDNA in the transition of BE to EAC. We analyzed WGS data from 206 patients in a cross-sectional surveillance cohort from Cambridge University UK, with biopsy-validated BE, including 42 patients who never developed high-grade dysplasia (HGD) or EAC during multi-year follow-ups, 25 patients with HGD, 51 patients with early-stage EAC, and 88 patients with late-stage EAC. ecDNA frequency increased between early-stage EAC (24%) and late-stage EAC (43%), suggesting continual ecDNA formation during cancer progression. We additionally analyzed WGS and histology data collected from multi-regional sampling of BE and EAC tissue at two time points from 80 BE patients in an observational case-control study at the Fred Hutchinson Cancer Center (FHCC), where 40 of the patients developed BE-associated EAC during the study and 40 did not. Among FHCC patients, 33% who developed EAC had at least one esophageal biopsy with ecDNA prior to, or at EAC diagnosis. In biopsies collected before cancer diagnosis, ecDNA presence in the biopsy was enriched among patients who later developed EAC compared to patients who did not. When linked to histology data, ecDNA-positive biopsies associated with worsened histological states before cancer diagnosis (p=0.015), and also at cancer diagnosis (p=0.037), with the pre-cancer timepoint being on average 2.1 years prior to cancer diagnosis. The ecDNAs had diverse collections of oncogenes and immunomodulatory genes, and showed higher copy number states than other focal amplification types. Furthermore, ecDNAs showed increasing copy number and structural complexity in more advanced disease stages highlighting their positive selection and ongoing structural evolution. In both FHCC and Cambridge cohorts, we found an association of TP53 loss with ecDNA formation demonstrating the role of prior genomic instability to form ecDNA. In total, our findings illustrate that oncogenic ecDNA can develop early in the malignant transformation, and that they are tied to worsening patient disease status. Citation Format: Jens Luebeck, Alvin W. Ng, Patricia C. Galipeau, Xiaohong Li, Annalise Katz-Summercorn, Hoon Kim, Sriganesh Jammula, Yudou He, Roel Verhaak, Carlo C. Maley, Ludmil B. Alexandrov, Rebecca C. Fitzgerald, Thomas G. Paulson, Howard Y. Chang, Sihan Wu, Vineet Bafna, Paul S. Mischel. Extrachromosomal DNA in the cancerous transformation of Barrett’s esophagus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3130.

Background Barrett’s oesophagus (BE) is the main risk factor for the development of oesophageal adenocarcinoma (OAC), yet few patients ever go on to progress to cancer. The acquisition of events during the metaplasia-dysplasia-cancer sequence is poorly characterised. We present a large, unbiased, multi-omics analysis of a cross-sectional cohort of pre-cancer samples, with the aim of providing a comprehensive insight into the diversity and molecular changes driving the disease to cancer. Methods We generated and integrated the genomic (50x), transcriptomic and epigenomic (850K EPIC array) landscapes of snap-frozen endoscopic biopsies from 146 patients with a range of outcomes (27 long-standing non-dysplastic; 12 prior to progression to dysplasia; 14 low-grade; 25 high-grade; 21 intramucosal carcinoma; 47 cases of BE taken adjacent to OAC) and 642 person years of follow-up. All biopsies were reviewed independently by 3 pathologists and had associated annotation with detailed clinical information. Results The total number of structural variants (SV) captured the most variance between samples. Complex SVs and LINE-1 retrotransposon activity were observed even before dysplasia had developed and increased with progression. Increasing SV burden was associated with chromothripsis (12%, 18/146) and breakage-fusion bridges (BFBs; 8%, 13/146). In more than 50% of these, the BFBs were in chromosome 17, harbouring the oncogenes ERBB2 and CDK12, for which expression was significantly higher. With progression there was increased expression of genes related to cell-cycle checkpoint, DNA repair and chromosomal instability, and the epigenetic silencing of genes in WNT-signalling and cell-cycle pathways. Conclusions Genomic complexity occurs very early in the natural history of BE and increasing genomic instability appears to tip the balance towards cancer. This may inform the potential for progression to cancer beyond the clinically discernible phenotype. Efforts to better understand the triggers for chromosomal breakages and rearrangements that underly progression will aid clinical prediction and prevention strategies.

Polyploidy is present in many cancer types and is increasingly recognized as an important factor in promoting chromosomal instability, genome evolution, and heterogeneity in cancer cells. However, the mechanisms that trigger polyploidy in cancer cells are largely unknown. In this study, we investigated the origin of polyploidy in esophageal adenocarcinoma (EAC), a highly heterogenous cancer, using a combination of genomics and cell biology approaches in EAC cell lines, organoids, and tumors. We found the EAC cells and organoids present specific mitotic defects consistent with problems in the attachment of chromosomes to the microtubules of the mitotic spindle. Time-lapse analyses confirmed that EAC cells have problems in congressing and aligning their chromosomes, which can ultimately culminate in mitotic slippage and polyploidy. Furthermore, whole-genome sequencing, RNA-seq, and quantitative immunofluorescence analyses revealed alterations in the copy number, expression, and cellular distribution of several proteins known to be involved in the mechanics and regulation of chromosome dynamics during mitosis. Together, these results provide evidence that an imbalance in the amount of proteins implicated in the attachment of chromosomes to spindle microtubules is the molecular mechanism underlying mitotic slippage in EAC. Our findings that the likely origin of polyploidy in EAC is mitotic failure caused by problems in chromosomal attachments not only improves our understanding of cancer evolution and diversification, but may also aid in the classification and treatment of EAC and possibly other highly heterogeneous cancers.

Identifying the origin of cancer Many cancers are classified on the basis of the organ or tissue from which they originated. However, identifying the specific cells and conditions that precede tumorigenesis can help us understand and better treat the resulting disease. Nowicki-Osuch et al . used a single-cell approach to investigate the cell of origin for Barrett’s esophagus (BE) and the mechanisms leading to the development of esophageal adenocarcinoma (EAC) (see the Perspective by Geboes and Hoorens). Analyses of healthy human esophageal tissues, mutational lineage tracing, and organoid models revealed that BE originates from the gastric cardia and that EAC arises from undifferentiated BE cells. This analysis provides a map of the transcriptional landscape of the healthy esophagus that can be compared with mouse models of disease. —LMZ

BACKGROUND & AIMS: Esophageal adenocarcinomas (EACs) 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 epigenetic analyses of BE and EAC tissues and combined these data with transcriptome and genomic data to identify mechanisms that control gene expression and genome integrity.METHODS: In a retrospective cohort study, we collected tissue samples and clinical data from 150 BE and 285 EAC cases from the Oesophageal Cancer Classification and Molecular Stratification consortium in the United Kingdom. We analyzed methylation profiles of all BE and EAC tissues and assigned them to subgroups using non-negative matrix factorization with k-means clustering. Data from whole-genome sequencing and transcriptome studies were then incorporated; we performed integrative methylation and RNA-sequencing analyses to identify genes that were suppressed with increased methylation in promoter regions. Levels of different immune cell types were computed using single-sample gene set enrichment methods. We derived 8 organoids from 8 EAC tissues and tested their sensitivity to different drugs.RESULTS: BE and EAC samples shared genome-wide methylation features, compared with normal tissues (esophageal, gastric, and duodenum; controls) from the same patients and grouped into 4 subtypes. Subtype 1 was characterized by DNA hypermethylation with a high mutation burden and multiple mutations in genes in cell cycle and receptor tyrosine signaling pathways. Subtype 2 was characterized by a gene expression pattern associated with metabolic processes (ATP synthesis and fatty acid oxidation) and lack methylation at specific binding sites for transcription factors; 83% of samples of this subtype were BE and 17% were EAC. The third subtype did not have changes in methylation pattern, compared with control tissue, but had a gene expression pattern that indicated immune cell infiltration; this tumor type was associated with the shortest time of patient survival. The fourth subtype was characterized by DNA hypomethylation associated with structure rearrangements, copy number alterations, with preferential amplification of CCNE1 (cells with this gene amplification have been reported to be sensitive to CDK2 inhibitors). Organoids with reduced levels of MGMT and CHFR expression were sensitive to temozolomide and taxane drugs.CONCLUSIONS: In a comprehensive integrated analysis of methylation, transcriptome, and genome profiles of more than 400 BE and EAC tissues, along with clinical data, we identified 4 subtypes that were associated with patient outcomes and potential responses to therapy.

Cancer remains a leading cause of morbidity and mortality worldwide. Its evolutionary nature and resultant complex interactions with the tumour micro-environment and the host immune system engender heterogeneity, make developing interventions difficult. Usually detected at the advanced stages of disease, metastatic cancer accounts for 90% of cancer-associated deaths. Therefore early detection of cancer, combined with current therapies, would have a significant impact on survival and treatment of this insidious disease. Epigenetic changes such as DNA methylation are some of the early events in carcinogenesis. Here, we report on a machine learning model that can classify 13 types of cancer as well as non-cancer tissue samples using only DNA methylome data, with an accuracy of 98.2%. We utilise the features identified by this model to develop a robust deep neural network that can generalise to independent data sets. We also demonstrate that the methylation associated genomic loci detected by the classifier are associated with genes involved in cancer, providing insights into the epigenomic regulation of carcinogenesis.

Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a-Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3-positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1-phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.

Chromatin modifications shape cell heterogeneity by activating and repressing defined sets of genes involved in cell proliferation, differentiation and development. Polycomb‐repressive complexes (PRCs) act synergistically during development and differentiation by maintaining transcriptional repression of common genes. PRC2 exerts this activity by catalysing H3K27 trimethylation. Here, we show that in the intestinal epithelium PRC2 is required to sustain progenitor cell proliferation and the correct balance between secretory and absorptive lineage differentiation programs. Using genetic models, we show that PRC2 activity is largely dispensable for intestinal stem cell maintenance but is strictly required for radiation‐induced regeneration by preventing Cdkn2a transcription. Combining these models with genomewide molecular analysis, we further demonstrate that preferential accumulation of secretory cells does not result from impaired proliferation of progenitor cells induced by Cdkn2a activation but rather from direct regulation of transcription factors responsible for secretory lineage commitment. Overall, our data uncover a dual role of PRC2 in intestinal homeostasis highlighting the importance of this repressive layer in controlling cell plasticity and lineage choices in adult tissues. ![][1] PRC2 exerts a dual role in intestinal homeostasis by controlling progenitor cell proliferation via repression of Cdkn2a and restricting secretory commitment by targeting key regulators of cell differentiation. [1]: /embed/graphic-1.gif

Background & Aims: Most patients with esophageal adenocarcinoma (EAC) present with de novo tumors. Although this could be due to inadequate screening strategies, the precise reason for this observation is not clear. We compared survival of patients with prevalent EAC with and without synchronous Barrett esophagus (BE) with intestinal metaplasia (IM) at the time of EAC diagnosis. Methods: Clinical data were studied using Cox proportional hazards regression to evaluate the effect of synchronous BE-IM on EAC survival independent of age, sex, TNM stage, and tumor location. We analyzed data from a cohort of patients with EAC from the Mayo Clinic (n=411; 203 with BE and IM) and a multicenter cohort from the United Kingdom (n=1417; 638 with BE and IM). Results: In the Mayo cohort, BE with IM had a reduced risk of death compared to patients without BE and IM (hazard ratio [HR] 0.44; 95% CI, 0.34–0.57; PConclusion: Two types of EAC can be characterized based on the presence or absence of BE. These findings could increase our understanding the etiology of EAC, and be used in management and prognosis of patients.

Heavy methyl Stable Isotope Labeling with Amino acids in Cell culture (hmSILAC) is a metabolic labeling strategy employed in proteomics to increase the confidence of global identification of methylated peptides by MS. However, to this day, the automatic and robust identification of heavy and light peak doublets from MS-raw data of hmSILAC experiments is a challenging task, for which the choice of computational methods is very limited. Here, hmSEEKER, a software designed to work downstream of a MaxQuant analysis for in-depth search of MS peak pairs that correspond to light and heavy methyl-peptide within MaxQuant-generated tables is described with good sensitivity and specificity. The software is written in Perl, and its code and user manual are freely available at Bitbucket (https://bit.ly/2scCT9u).

Background & Aims MicroRNA (miRNA) is highly stable in biospecimens and provides tissue-specific profiles, making it a useful biomarker of carcinogenesis. We aimed to discover a set of miRNAs that could accurately discriminate Barrett’s esophagus (BE) from normal esophageal tissue and to test its diagnostic accuracy when applied to samples collected by a noninvasive esophageal cell sampling device. Methods We analyzed miRNA expression profiles of 2 independent sets of esophageal biopsy tissues collected during endoscopy from 38 patients with BE and 26 patients with normal esophagus (controls) using Agilent microarray and Nanostring nCounter assays. Consistently up-regulated miRNAs were quantified by real-time polymerase chain reaction in esophageal tissues collected by Cytosponge from patients with BE vs without BE. miRNAs were expressed from plasmids and antisense oligonucleotides were expressed in normal esophageal squamous cells; effects on proliferation and gene expression patterns were analyzed. Results We identified 15 miRNAs that were significantly up-regulated in BE vs control tissues. Of these, 11 (MIR215, MIR194, MIR 192, MIR196a, MIR199b, MIR10a, MIR145, MIR181a, MIR30a, MIR7, and MIR199a) were validated in Cytosponge samples. The miRNAs with the greatest increases in BE tissues (7.9-fold increase in expression or more, P < .0001: MIR196a, MIR192, MIR194, and MIR215) each identified BE vs control tissues with area under the curve (AUC) values of 0.82 or more. We developed an optimized multivariable logistic regression model, based on expression levels of 6 miRNAs (MIR7, MIR30a, MIR181a, MIR192, MIR196a, and MIR199a), that identified patients with BE with an AUC value of 0.89, 86.2% sensitivity, and 91.6% specificity. Expression level of MIR192, MIR196a, MIR199a, combined that of trefoil factor 3, identified patients with BE with an AUC of 0.93, 93.1% sensitivity, and 93.7% specificity. Hypomethylation was observed in the promoter region of the highly up-regulated cluster MIR192–MIR194. Overexpression of these miRNAs in normal esophageal squamous cells increased their proliferation, via GRHL3 and PTEN signaling. Conclusions In analyses of miRNA expression patterns of BE vs non-BE tissues, we identified a profile that can identify Cytosponge samples from patients with BE with an AUC of 0.93. Expression of MIR194 is increased in BE samples via epigenetic mechanisms that might be involved in BE pathogenesis., Graphical abstract

The Polycomb repressor complex 2 (PRC2) is composed of the core subunits Ezh1/2, Suz12, and Eed, and it mediates all di- and tri-methylation of histone H3 at lysine 27 in higher eukaryotes. However, little is known about how the catalytic activity of PRC2 is regulated to demarcate H3K27me2 and H3K27me3 domains across the genome. To address this, we mapped the endogenous interactomes of Ezh2 and Suz12 in embryonic stem cells (ESCs), and we combined this with a functional screen for H3K27 methylation marks. We found that Nsd1-mediated H3K36me2 co-locates with H3K27me2, and its loss leads to genome-wide expansion of H3K27me3. These increases in H3K27me3 occurred at PRC2/PRC1 target genes and as de novo accumulation within what were previously broad H3K27me2 domains. Our data support a model in which Nsd1 is a key modulator of PRC2 function required for regulating the demarcation of genome-wide H3K27me2 and H3K27me3 domains in ESCs.

4063 Background: Anti-PD-1 therapy modestly improves survival in chemorefractory OGA. Combining PD-1 blockade with novel checkpoint inhibitors, T-cell co-stimulatory molecules, or myeloid suppressors could enhance PD-1 inhibition. Herein, we explore the landscape of known targetable immune markers in non-Asian OGA. Methods: OGA patient biopsies were prospectively collected and clinically annotated from 19 UK cancer centres (Oesophageal Cancer Clinical and Molecular Stratification – OCCAMs network). Genomic (WGS) and transcriptomic (bulk-RNA seq) data were generated using Illumina and processed using a validated in-house pipeline (Frankell, Nat Genetics 2019). Gene expression was computed in transcripts per kilobase. Using unsupervised clustering patient clusters were selected according to the expression of gene targets for immune therapy - PD-L1, LAG3, TIM3, TIGIT, ICOS, CCR2, CCR5, CXCR4, and CSF1R. Immune cell infiltration was extrapolated using GSVA gene set enrichment analysis. Results: RNAseq data were available for 251 patients; 96% had operable tumours (Stage I: 6%, II: 63%, III: 22%, IV: 4%). In untreated patients (n = 156) 3 subgroups were identified: immune low (83, 53%) with low level expression of all 9 markers, immune high (14, 9%) with high expression of all or majority of markers and intermediate (59, 38%) with heterogenous marker expression. Clinicopathological variables (sex, age, smoking, tumour location (gastric/GEJ/esophagus) and tumour regression grade) were similarly distributed across subgroups. In a cohort of 114 patients with matched WGS and RNAseq data tumour mutation burden was not different between subgroups. In post-chemotherapy biopsies (n = 95) a similar co-expression pattern was observed. Gene enrichment analysis supported infiltration by cells of innate and adaptive immune system in immune high patients. Neoantigen results, phenotypic immunohistochemistry and optimised survival outcomes according to lymphoid and myeloid target expression will be presented. Conclusions: High level co-expression of immune regulatory targets in OGA patients may limit the efficacy of anti-PD-1 monotherapy. Combination immune directed therapies may be required in this patient group.

H3K27me3 is deposited at promoters by the preferential association of Polycomb repressive complex 2 (PRC2) with CpG-rich DNA elements regulating development by repressing gene transcription. H3K27 is also present in mono- and dimethylated states; however, the functional roles of H3K27me1 and H3K27me2 deposition remain poorly characterized. Here, we show that PRC2 activity is not only associated with H3K27me3 but also regulates all forms of H3K27 methylation in a spatially defined manner, contributing to different genomic functions in mouse embryonic stem cells. H3K27me1 accumulates within transcribed genes, promotes transcription, and is regulated by Setd2-dependent H3K36me3 deposition. Contrarily, H3K27me2 is present on approximately 70% of total histone H3 and is distributed in large chromatin domains, exerting protective functions by preventing firing of non-cell-type-specific enhancers. Considering that only 5%-10% of deregulated genes in PRC2-deficient cells are direct H3K27me3 targets, our data support an active role for all H3K27 methylated forms in regulating transcription and determining cell identity.

The Polycomb complex PRC1 is a gatekeeper of leukemic cell identity in the presence of distinct oncogenic signals., Leukemia is a complex heterogeneous disease often driven by the expression of oncogenic fusion proteins with different molecular and biochemical properties. Whereas several fusion proteins induce leukemogenesis by activating Hox gene expression (Hox-activating fusions), others impinge on different pathways that do not involve the activation of Hox genes (non–Hox-activating fusions). It has been postulated that one of the main oncogenic properties of the HOXA9 transcription factor is its ability to control the expression of the p16/p19 tumor suppressor locus (Cdkn2a), thereby compensating Polycomb-mediated repression, which is dispensable for leukemias induced by Hox-activating fusions. We show, by genetically depleting the H2A ubiquitin ligase subunits of the Polycomb repressive complex 1 (PRC1), Ring1a and Ring1b, that Hoxa9 activation cannot repress Cdkn2a expression in the absence of PRC1 and its dependent deposition of H2AK119 monoubiquitination (H2AK119Ub). This demonstrates the essential role of PRC1 activity in supporting the oncogenic potential of Hox-activating fusion proteins. By combining genetic tools with genome-wide location and transcription analyses, we further show that PRC1 activity is required for the leukemogenic potential of both Hox-activating and non–Hox-activating fusions, thus preventing the differentiation of leukemic cells independently of the expression of the Cdkn2a locus. Overall, our results genetically demonstrate that PRC1 activity and the deposition of H2AK119Ub are critical factors that maintain the undifferentiated identity of cancer cells, positively sustaining the progression of different types of leukemia.

Polycomb repressive complexes (PRCs) are among the most important gatekeepers of establishing and maintaining cell identity in metazoans. PRC1, which plays a dominant role in this context, executes its functions via multiple subcomplexes, which all contribute to H2AK119 mono-ubiquitination (H2Aubq). Despite our comprehensive knowledge of PRC1-dependent H2Aubq in embryonic stem cells and during early development, its role in adult stem cells still remains poorly characterized. Here we show that PRC1 activity is required for the integrity of the intestinal epithelium, regulating stem cell self-renewal via a cell-autonomous mechanism that is independent from Cdkn2a expression. By dissecting the PRC1-dependent transcription program in intestinal stem cells, we demonstrate that PRC1 represses a large number of non-lineage-specific transcription factors that directly affect beta-catenin/Tcf transcriptional activity. Our data reveal that PRC1 preserves Wnt/beta-catenin activity in adult stem cells to maintain intestinal homeostasis and supports tumor formation induced by the constitutive activation of this pathway.

Additional file 1. Contains two supplementary figures and corresponding legends: Figure S1 provides a graphical description of the quantification workflow. Figure S2 provides a comparison of differences in different modes of quantification, annotation of differentially regulated ROI and quantification of different ChIPs in Suz12 binding regions.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) activity is essential for embryonic stem cell (ESC) viability and mouse development. Ogt is present both in the cytoplasm and the nucleus of different cell types and catalyzes serine and threonine glycosylation. We have characterized the biochemical features of nuclear Ogt and identified the ten-eleven translocation (TET) proteins Tet1 and Tet2 as stable partners of Ogt in the nucleus of ESCs. We show at a genome-wide level that Ogt preferentially associates with Tet1 to genes promoters in close proximity of CpG-rich transcription start sites. These regions are characterized by low levels of DNA modification, suggesting a link between Tet1 and Ogt activities in regulating CpG island methylation. Finally, we show that Tet1 is required for binding of Ogt to chromatin affecting Tet1 activity. Taken together, our data characterize how O-GlcNAcylation is recruited to chromatin and interacts with the activity of 5-methylcytosine hydroxylases.

Background In epigenetic research, both the increasing ease of high-throughput sequencing and a greater interest in genome-wide studies have resulted in an exponential flooding of epigenetic-related data in public domain. This creates an opportunity for exploring data outside the limits of any specific query-centred study. Such data have to undergo standard primary analyses that are accessible with multiple well-stabilized programs. Further downstream analyses, such as genome-wide comparative, correlative and quantitative analyses, are critical in deciphering key biological features. However, these analyses are only accessible for computational researchers and completely lack platforms capable of handling, analysing and linking multiple interdisciplinary datasets with efficient analytical methods. Results Here, we present EpiMINE, a program for mining epigenomic data. It is a user-friendly, stand-alone computational program designed to support multiple datasets, for performing genome-wide correlative and quantitative analysis of ChIP-seq and RNA-seq data. Using data available from the ENCODE project, we illustrated several features of EpiMINE through different biological scenarios to show how easy some known observations can be verified. These results highlight how these approaches can be helpful in identifying novel biological features. Conclusions EpiMINE performs different kinds of genome-wide quantitative and correlative analyses, using ChIP-seq- and RNA-seq-related datasets. Its framework enables it to be used by both experimental and computational researchers. EpiMINE can be downloaded from https://sourceforge.net/projects/epimine/. Electronic supplementary material The online version of this article (doi:10.1186/s13072-016-0095-z) contains supplementary material, which is available to authorized users.