Shona MacRae

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.

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

BACKGROUND Barretts oesophagus predisposes to adenocarcinoma. However, most patients with Barretts 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 Barretts 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 Barretts 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 Barretts 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 Barretts oesophagus. FUNDING Cancer Research UK.

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.

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 ZNF569methylation have promise as diagnostic biomarkers for Barrett’s oesophagus when used in combination with a simple and cost effective non-endoscopic cell collection device.

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. Our understanding of genetic events which drive EAC development is limited and there are few molecular biomarkers for prognostication or therapeutics. We have accumulated a cohort of 551 genomically characterised EACs (73% WGS and 27% WES) with clinical annotation and matched RNA-seq. Using a variety of driver gene detection methods we discover 65 EAC drivers (66% novel) and describe mutation and CNV types with specific functional impact. We identify a mean of 3.7 driver events per case derived almost equally from copy number events and mutations. We compare driver mutation rates to the exome-wide mutational excess calculated using Non-synonymous vs Synonymous mutation rates (dNdS). We see mutual exclusivity or co-occurrence of events within and between a number of EAC pathways (GATA factors, Core Cell cycle genes, TP53 regulators and the SWI/SNF complex) suggestive of important functional relationships. These driver variants correlate with tumour differentiation, sex and prognosis. Poor prognostic indicators (SMAD4, GATA4) are verified in independent cohorts with significant predictive value. Over 50% of EACs contain sensitising events for CDK4/6 inhibitors which are highly correlated with clinically relevant sensitivity in a panel EAC cell lines.

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.Esophageal adenocarcinoma (EAC) has a poor 5-year survival rate and lacks robust preclinical models for use in research. Here, the authors show that newly derived organoids recapitulate the transcriptomic, genetic, and morphological landscape of the primary EAC tumors and provide a platform to test drug sensitivity and study tumor clonality.

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.

Erratum: Corrigendum: Mutational signatures in esophageal adenocarcinoma define etiologically distinct subgroups with therapeutic relevance

Nat. Genet.; 10.1038/ng.3659; corrected online 19 September 2016 In the version of this article initially published online, the mutation signature illustrations for S1 and S2 in Figure 3a were switched. Additionally, in the Online Methods, the text originally stated that structural variants were called using BWA-MEM, when it should have stated that these were called using BWA.

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

Corrigendum: A comparative analysis of whole genome sequencing of esophageal adenocarcinoma preand post-chemotherapy Ayesha Noorani, Jan Bornschein, Andy G. Lynch, Maria Secrier, Achilleas Achilleos, Matthew Eldridge, Lawrence Bower, Jamie M.J. Weaver, Jason Crawte, Chin-Ann Ong, Nicholas Shannon, Shona MacRae, Nicola Grehan, Barbara Nutzinger, Maria O’Donovan, Richard Hardwick, Simon Tavaré, Rebecca C. Fitzgerald, on behalf of the Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) Consortium