Genomic evidence supports a clonal diaspora model for metastases of esophageal adenocarcinoma.

The poor outcomes in esophageal adenocarcinoma (EAC) prompted us to interrogate the pattern and timing of metastatic spread. Whole-genome sequencing and phylogenetic analysis of 388 samples across 18 individuals with EAC showed, in 90% of patients, that multiple subclones from the primary tumor spread very rapidly from the primary site to form multiple metastases, including lymph nodes and distant tissues-a mode of dissemination that we term 'clonal diaspora'. Metastatic subclones at autopsy were present in tissue and blood samples from earlier time points. These findings have implications for our understanding and clinical evaluation of EAC.

Transcriptomic profiling reveals three molecular phenotypes of adenocarcinoma at the gastroesophageal junction.

Cancers occurring at the gastroesophageal junction (GEJ) are classified as predominantly esophageal or gastric, which is often difficult to decipher. We hypothesized that the transcriptomic profile might reveal molecular subgroups which could help to define the tumor origin and behavior beyond anatomical location. The gene expression profiles of 107 treatment-naïve, intestinal type, gastroesophageal adenocarcinomas were assessed by the Illumina-HTv4.0 beadchip. Differential gene expression (limma), unsupervised subgroup assignment (mclust) and pathway analysis (gage) were undertaken in R statistical computing and results were related to demographic and clinical parameters. Unsupervised assignment of the gene expression profiles revealed three distinct molecular subgroups, which were not associated with anatomical location, tumor stage or grade (p > 0.05). Group 1 was enriched for pathways involved in cell turnover, Group 2 was enriched for metabolic processes and Group 3 for immune-response pathways. Patients in group 1 showed the worst overall survival (p = 0.019). Key genes for the three subtypes were confirmed by immunohistochemistry. The newly defined intrinsic subtypes were analyzed in four independent datasets of gastric and esophageal adenocarcinomas with transcriptomic data available (RNAseq data: OCCAMS cohort, n = 158; gene expression arrays: Belfast, n = 63; Singapore, n = 191; Asian Cancer Research Group, n = 300). The subgroups were represented in the independent cohorts and pooled analysis confirmed the prognostic effect of the new subtypes. In conclusion, adenocarcinomas at the GEJ comprise three distinct molecular phenotypes which do not reflect anatomical location but rather inform our understanding of the key pathways expressed.

Immune activation by DNA damage predicts response to chemotherapy and survival in oesophageal adenocarcinoma.

OBJECTIVE: Current strategies to guide selection of neoadjuvant therapy in oesophageal adenocarcinoma (OAC) are inadequate. We assessed the ability of a DNA damage immune response (DDIR) assay to predict response following neoadjuvant chemotherapy in OAC. DESIGN: Transcriptional profiling of 273 formalin-fixed paraffin-embedded prechemotherapy endoscopic OAC biopsies was performed. All patients were treated with platinum-based neoadjuvant chemotherapy and resection between 2003 and 2014 at four centres in the Oesophageal Cancer Clinical and Molecular Stratification consortium. CD8 and programmed death ligand 1 (PD-L1) immunohistochemical staining was assessed in matched resection specimens from 126 cases. Kaplan-Meier and Cox proportional hazards regression analysis were applied according to DDIR status for recurrence-free survival (RFS) and overall survival (OS). RESULTS: A total of 66 OAC samples (24%) were DDIR positive with the remaining 207 samples (76%) being DDIR negative. DDIR assay positivity was associated with improved RFS (HR: 0.61; 95% CI 0.38 to 0.98; p=0.042) and OS (HR: 0.52; 95% CI 0.31 to 0.88; p=0.015) following multivariate analysis. DDIR-positive patients had a higher pathological response rate (p=0.033), lower nodal burden (p=0.026) and reduced circumferential margin involvement (p=0.007). No difference in OS was observed according to DDIR status in an independent surgery-alone dataset.DDIR-positive OAC tumours were also associated with the presence of CD8+ lymphocytes (intratumoural: p<0.001; stromal: p=0.026) as well as PD-L1 expression (intratumoural: p=0.047; stromal: p=0.025). CONCLUSION: The DDIR assay is strongly predictive of benefit from DNA-damaging neoadjuvant chemotherapy followed by surgical resection and is associated with a proinflammatory microenvironment in OAC.

The landscape of selection in 551 esophageal adenocarcinomas defines genomic biomarkers for the clinic.

Esophageal adenocarcinoma (EAC) is a poor-prognosis cancer type with rapidly rising incidence. Understanding of the genetic events driving EAC development is limited, and there are few molecular biomarkers for prognostication or therapeutics. Using a cohort of 551 genomically characterized EACs with matched RNA sequencing data, we discovered 77 EAC driver genes and 21 noncoding driver elements. We identified a mean of 4.4 driver events per tumor, which were derived more commonly from mutations than copy number alterations, and compared the prevelence of these mutations to the exome-wide mutational excess calculated using non-synonymous to synonymous mutation ratios (dN/dS). We observed mutual exclusivity or co-occurrence of events within and between several dysregulated EAC pathways, a result suggestive of strong functional relationships. Indicators of poor prognosis (SMAD4 and GATA4) were verified in independent cohorts with significant predictive value. Over 50% of EACs contained sensitizing events for CDK4 and CDK6 inhibitors, which were highly correlated with clinically relevant sensitivity in a panel of EAC cell lines and organoids.

Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics.

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.

Selection and Application of Tissue microRNAs for Nonendoscopic Diagnosis of Barrett's Esophagus.

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.

Methylation panel is a diagnostic biomarker for Barrett's oesophagus in endoscopic biopsies and non-endoscopic cytology specimens.

OBJECTIVE: Barrett's oesophagus is a premalignant condition that occurs in the context of gastro-oesophageal reflux. However, most Barrett's cases are undiagnosed because of reliance on endoscopy. We have developed a non-endoscopic tool: the Cytosponge, which when combined with trefoil factor 3 immunohistochemistry, can diagnose Barrett's oesophagus. We investigated whether a quantitative methylation test that is not reliant on histopathological analysis could be used to diagnose Barrett's oesophagus. DESIGN: Differentially methylated genes between Barrett's and normal squamous oesophageal biopsies were identified from whole methylome data and confirmed using MethyLight PCR in biopsy samples of squamous oesophagus, gastric cardia and Barrett's oesophagus. Selected genes were then tested on Cytosponge BEST2 trial samples comprising a pilot cohort (n=20 cases, n=10 controls) and a validation cohort (n=149 cases, n=129 controls). RESULTS: Eighteen genes were differentially methylated in patients with Barrett'soesophagus compared with squamous controls. Hypermethylation of TFPI2, TWIST1, ZNF345 and ZNF569 was confirmed in Barrett's biopsies compared with biopsies from squamous oesophagus and gastric cardia (p<0.05). When tested in Cytosponge samples, these four genes were hypermethylated in patients with Barrett's oesophagus compared with patients with reflux symptoms (p<0.001). The optimum biomarker to diagnose Barrett's oesophagus was TFPI2 with a sensitivity and specificity of 82.2% and 95.7%, respectively. CONCLUSION: TFPI2, TWIST1, ZNF345 and ZNF569 methylation have promise as diagnostic biomarkers for Barrett's oesophagus when used in combination with a simple and cost effective non-endoscopic cell collection device.

A comparative analysis of whole genome sequencing of esophageal adenocarcinoma pre- and post-chemotherapy.

The scientific community has avoided using tissue samples from patients that have been exposed to systemic chemotherapy to infer the genomic landscape of a given cancer. Esophageal adenocarcinoma is a heterogeneous, chemoresistant tumor for which the availability and size of pretreatment endoscopic samples are limiting. This study compares whole-genome sequencing data obtained from chemo-naive and chemo-treated samples. The quality of whole-genomic sequencing data is comparable across all samples regardless of chemotherapy status. Inclusion of samples collected post-chemotherapy increased the proportion of late-stage tumors. When comparing matched pre- and post-chemotherapy samples from 10 cases, the mutational signatures, copy number, and SNV mutational profiles reflect the expected heterogeneity in this disease. Analysis of SNVs in relation to allele-specific copy-number changes pinpoints the common ancestor to a point prior to chemotherapy. For cases in which pre- and post-chemotherapy samples do show substantial differences, the timing of the divergence is near-synchronous with endoreduplication. Comparison across a large prospective cohort (62 treatment-naive, 58 chemotherapy-treated samples) reveals no significant differences in the overall mutation rate, mutation signatures, specific recurrent point mutations, or copy-number events in respect to chemotherapy status. In conclusion, whole-genome sequencing of samples obtained following neoadjuvant chemotherapy is representative of the genomic landscape of esophageal adenocarcinoma. Excluding these samples reduces the material available for cataloging and introduces a bias toward the earlier stages of cancer.

Risk stratification of Barrett's oesophagus using a non-endoscopic sampling method coupled with a biomarker panel: a cohort study.

BACKGROUND: Barrett's oesophagus predisposes to adenocarcinoma. However, most patients with Barrett's oesophagus will not progress and endoscopic surveillance is invasive, expensive, and fraught by issues of sampling bias and the subjective assessment of dysplasia. We investigated whether a non-endoscopic device, the Cytosponge, could be coupled with clinical and molecular biomarkers to identify a group of patients with low risk of progression suitable for non-endoscopic follow-up. METHODS: In this multicentre cohort study (BEST2), patients with Barrett's oesophagus underwent the Cytosponge test before their surveillance endoscopy. We collected clinical and demographic data and tested Cytosponge samples for a molecular biomarker panel including three protein biomarkers (P53, c-Myc, and Aurora kinase A), two methylation markers (MYOD1 and RUNX3), glandular atypia, and TP53 mutation status. We used a multivariable logistic regression model to compute the conditional probability of dysplasia status. We selected a simple model with high classification accuracy and applied it to an independent validation cohort. The BEST2 study is registered with ISRCTN, number 12730505. FINDINGS: The discovery cohort consisted of 468 patients with Barrett's oesophagus and intestinal metaplasia. Of these, 376 had no dysplasia and 22 had high-grade dysplasia or intramucosal adenocarcinoma. In the discovery cohort, a model with high classification accuracy consisted of glandular atypia, P53 abnormality, and Aurora kinase A positivity, and the interaction of age, waist-to-hip ratio, and length of the Barrett's oesophagus segment. 162 (35%) of 468 of patients fell into the low-risk category and the probability of being a true non-dysplastic patient was 100% (99% CI 96-100) and the probability of having high-grade dysplasia or intramucosal adenocarcinoma was 0% (0-4). 238 (51%) of participants were classified as of moderate risk; the probability of having high-grade dysplasia was 14% (9-21). 58 (12%) of participants were classified as high-risk; the probability of having non-dysplastic endoscopic biopsies was 13% (5-27), whereas the probability of having high-grade dysplasia or intramucosal adenocarcinoma was 87% (73-95). In the validation cohort (65 patients), 51 were non-dysplastic and 14 had high-grade dysplasia. In this cohort, 25 (38%) of 65 patients were classified as being low-risk, and the probability of being non-dysplastic was 96·0% (99% CI 73·80-99·99). The moderate-risk group comprised 27 non-dysplastic and eight high-grade dysplasia cases, whereas the high-risk group (8% of the cohort) had no non-dysplastic cases and five patients with high-grade dysplasia. INTERPRETATION: A combination of biomarker assays from a single Cytosponge sample can be used to determine a group of patients at low risk of progression, for whom endoscopy could be avoided. This strategy could help to avoid overdiagnosis and overtreatment in patients with Barrett's oesophagus. FUNDING: Cancer Research UK.

Mutational signatures in esophageal adenocarcinoma define etiologically distinct subgroups with therapeutic relevance.

Esophageal adenocarcinoma (EAC) has a poor outcome, and targeted therapy trials have thus far been disappointing owing to a lack of robust stratification methods. Whole-genome sequencing (WGS) analysis of 129 cases demonstrated 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 RTK inhibitor (RTKi) therapy might be required, as we demonstrate in vitro. However, mutational signatures showed three distinct molecular subtypes with potential therapeutic relevance, which we verified in an independent cohort (n = 87): (i) enrichment 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; and (iii) C>A/T mutational pattern with evidence of an aging imprint. These subtypes could be ascertained using a clinically applicable sequencing strategy (low coverage) as a basis for therapy selection.

The human squamous oesophagus has widespread capacity for clonal expansion from cells at diverse stages of differentiation.

OBJECTIVE: Knowledge of the cellular mechanisms involved in homeostasis of human squamous oesophagus in the steady state and following chronic injury is limited. We aimed to better understand these mechanisms by using a functional 3D approach. DESIGN: Proliferation, mitosis and the expression of progenitor lineage markers were assessed in normal squamous oesophagus from 10 patients by immunofluorescence on 3D epithelial whole mounts. Cells expressing differential levels of epithelial and progenitor markers were isolated using flow cytometry sorting and characterised by qPCR and IF. Their self-renewing potential was investigated by colony forming cells assays and in vitro organotypic culture models. RESULTS: Proliferation and mitotic activity was highest in the interpapillary basal layer and decreased linearly towards the tip of the papilla (p<0.0001). The orientation of mitosis was random throughout the basal layer, and asymmetric divisions were not restricted to specific cell compartments. Cells sorted into distinct populations based on the expression of epithelial and progenitor cell markers (CD34 and EpCAM) showed no difference in self-renewal in 2D culture, either as whole populations or as single cells. In 3D organotypic cultures, all cell subtypes were able to recapitulate the architecture of the tissue of origin and the main factor determining the success of the 3D culture was the number of cells plated, rather than the cell type. CONCLUSIONS: Oesophageal epithelial cells demonstrate remarkable plasticity for self-renewal. This situation could be viewed as an ex vivo wounding response and is compatible with recent findings in murine models.

The genetics of virus particle shape in equine influenza A virus.

BACKGROUND: Many human strains of influenza A virus produce highly pleomorphic virus particles that at the extremes can be approximated as either spheres of around 100 nm diameter or filaments of similar cross-section but elongated to lengths of many microns. The role filamentous virions play in the virus life cycle remains enigmatic. OBJECTIVES/METHODS: Here, we set out to define the morphology and genetics of virus particle shape in equine influenza A virus, using reverse genetics and microscopy of infected cells. RESULTS AND CONCLUSIONS: The majority of H3N8 strains tested were found to produce filamentous virions, as did the prototype H7N7 A/eq/Prague/56 strain. The exception was the prototype H3N8 isolate, A/eq/Miami/63. Reassortment of equine influenza virus M genes from filamentous and non-filamentous strains into the non-filamentous human virus A/PR/8/34 confirmed that segment 7 is a major determinant of particle shape. Sequence analysis identified three M1 amino acid polymorphisms plausibly associated with determining virion morphology, and the introduction of these changes into viruses confirmed the importance of two: S85N and N231D. However, while either change alone affected filament production, the greatest effect was seen when the polymorphisms were introduced in conjunction. Thus, influenza A viruses from equine hosts also produce filamentous virions, and the major genetic determinants are set by the M1 protein. However, the precise sequence determinants are different to those previously identified in human or porcine viruses.

Equine influenza: a review of an unpredictable virus.

This review discusses some of the challenges still faced in the control of equine influenza virus H3N8 infection. A widespread outbreak of equine influenza in the United Kingdom during 2003 in vaccinated Thoroughbred racehorses challenged the current dogma on vaccine strain selection. Furthermore, several new developments in the first decade of the 21st century, including transmission to and establishment in dogs, a presumed influenza-associated encephalopathy in horses and an outbreak of equine influenza in Australia, serve as a reminder of the unpredictable nature of influenza viruses. The application of newly available techniques described in this review may further elucidate some of the viral factors that underlie recent events and provide the tools to better evaluate when vaccine strains should be updated.

Dynamics of influenza virus infection and pathology.

A key question in pandemic influenza is the relative roles of innate immunity and target cell depletion in limiting primary infection and modulating pathology. Here, we model these interactions using detailed data from equine influenza virus infection, combining viral and immune (type I interferon) kinetics with estimates of cell depletion. The resulting dynamics indicate a powerful role for innate immunity in controlling the rapid peak in virus shedding. As a corollary, cells are much less depleted than suggested by a model of human influenza based only on virus-shedding data. We then explore how differences in the influence of viral proteins on interferon kinetics can account for the observed spectrum of virus shedding, immune response, and influenza pathology. In particular, induction of high levels of interferon ("cytokine storms"), coupled with evasion of its effects, could lead to severe pathology, as hypothesized for some fatal cases of influenza.

Antigenic and genetic variations in European and North American equine influenza virus strains (H3N8) isolated from 2006 to 2007.

Equine influenza virus (EIV) surveillance is important in the management of equine influenza. It provides data on circulating and newly emerging strains for vaccine strain selection. To this end, antigenic characterisation by haemaggluttination inhibition (HI) assay and phylogenetic analysis was carried out on 28 EIV strains isolated in North America and Europe during 2006 and 2007. In the UK, 20 viruses were isolated from 28 nasopharyngeal swabs that tested positive by enzyme-linked immunosorbent assay. All except two of the UK viruses were characterised as members of the Florida sublineage with similarity to A/eq/Newmarket/5/03 (clade 2). One isolate, A/eq/Cheshire/1/06, was characterised as an American lineage strain similar to viruses isolated up to 10 years earlier. A second isolate, A/eq/Lincolnshire/1/07 was characterised as a member of the Florida sublineage (clade 1) with similarity to A/eq/Wisconsin/03. Furthermore, A/eq/Lincolnshire/1/06 was a member of the Florida sublineage (clade 2) by haemagglutinin (HA) gene sequence, but appeared to be a member of the Eurasian lineage by the non-structural gene (NS) sequence suggesting that reassortment had occurred. A/eq/Switzerland/P112/07 was characterised as a member of the Eurasian lineage, the first time since 2005 that isolation of a virus from this lineage has been reported. Seven viruses from North America were classified as members of the Florida sublineage (clade 1), similar to A/eq/Wisconsin/03. In conclusion, a variety of antigenically distinct EIVs continue to circulate worldwide. Florida sublineage clade 1 viruses appear to predominate in North America, clade 2 viruses in Europe.

Transmission of equine influenza virus to English foxhounds.

We retrospectively demonstrated that an outbreak of severe respiratory disease in a pack of English foxhounds in the United Kingdom in September 2002 was caused by an equine influenza A virus (H3N8). We also demonstrated that canine respiratory tissue possesses the relevant receptors for infection with equine influenza virus.