A non-endoscopic device to sample the oesophageal microbiota: a case-control study.

BACKGROUND: The strongest risk factor for oesophageal adenocarcinoma is reflux disease, and the rising incidence of this coincides with the eradication of Helicobacter pylori, both of which might alter the oesophageal microbiota. We aimed to profile the microbiota at different stages of Barrett's carcinogenesis and investigate the Cytosponge as a minimally invasive tool for sampling the oesophageal microbiota. METHODS: In this case-control study, 16S rRNA gene amplicon sequencing was done on 210 oesophageal samples from 86 patients representing the Barrett's oesophagus progression sequence (normal squamous controls [n=20], non-dysplastic [n=24] and dysplastic Barrett's oesophagus [n=23], and oesophageal adenocarcinoma [n=19]), relevant negative controls, and replicates on the Illumina MiSeq platform. Samples were taken from patients enrolled in the BEST2 study at five UK hospitals and the OCCAMS study at six UK hospitals. We compared fresh frozen tissue, fresh frozen endoscopic brushings, and the Cytosponge device for microbial DNA yield (qPCR), diversity, and community composition. FINDINGS: There was decreased microbial diversity in oesophageal adenocarcinoma tissue compared with tissue from healthy control patients as measured by the observed operational taxonomic unit (OTU) richness (p=0·0012), Chao estimated total richness (p=0·0004), and Shannon diversity index (p=0·0075). Lactobacillus fermentum was enriched in oesophageal adenocarcinoma (p=0·028), and lactic acid bacteria dominated the microenvironment in seven (47%) of 15 cases of oesophageal adenocarcinoma. Comparison of oesophageal sampling methods showed that the Cytosponge yielded more than ten-times higher quantities of microbial DNA than did endoscopic brushes or biopsies using quantitative PCR (p<0·0001). The Cytosponge samples contained the majority of taxa detected in biopsy and brush samples, but were enriched for genera from the oral cavity and stomach, including Fusobacterium, Megasphaera, Campylobacter, Capnocytophaga, and Dialister. The Cytosponge detected decreased microbial diversity in patients with high-grade dysplasia in comparison to control patients, as measured by the observed OTU richness (p=0·0147), Chao estimated total richness (p=0·023), and Shannon diversity index (p=0·0085). INTERPRETATION: Alterations in microbial communities occur in the lower oesophagus in Barrett's carcinogenesis, which can be detected at the pre-invasive stage of high-grade dysplasia with the novel Cytosponge device. Our findings are potentially applicable to early disease detection, and future test development should focus on longitudinal sampling of the microbiota to monitor for changes in microbial diversity in a larger cohort of patients. FUNDING: Cancer Research UK, National Institute for Health Research, Medical Research Council, Wellcome Trust, The Scottish Government (RESAS).Cancer Research UK, National Institute for Health Research, Medical Research Council, and Wellcome Trust.This is the author accepted manuscript. The final version is available from Elsevier via https://doi.org/10.1016/S2468-1253(16)30086-

Whole-genome sequencing of nine esophageal adenocarcinoma cell lines.

Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines-ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4-all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC.This work was funded by an MRC Programme Grant to R.C.F. and a Cancer Research UK grant to PAWE. The pipeline for mutation calling is funded by Cancer Research UK as part of the International Cancer Genome Consortium. G.C. is a National Institute for Health Research Lecturer as part of a NIHR professorship grant to R.C.F. AGL is supported by a Cancer Research UK programme grant (C14303/A20406) to Simon Tavaré and the European Commission through the Horizon 2020 project SOUND (Grant Agreement no. 633974)

Impact of mutations in Toll-like receptor pathway genes on esophageal carcinogenesis.

Esophageal adenocarcinoma (EAC) develops in an inflammatory microenvironment with reduced microbial diversity, but mechanisms for these influences remain poorly characterized. We hypothesized that mutations targeting the Toll-like receptor (TLR) pathway could disrupt innate immune signaling and promote a microenvironment that favors tumorigenesis. Through interrogating whole genome sequencing data from 171 EAC patients, we showed that non-synonymous mutations collectively affect the TLR pathway in 25/171 (14.6%, PathScan p = 8.7x10-5) tumors. TLR mutant cases were associated with more proximal tumors and metastatic disease, indicating possible clinical significance of these mutations. Only rare mutations were identified in adjacent Barrett's esophagus samples. We validated our findings in an external EAC dataset with non-synonymous TLR pathway mutations in 33/149 (22.1%, PathScan p = 0.05) tumors, and in other solid tumor types exposed to microbiomes in the COSMIC database (10,318 samples), including uterine endometrioid carcinoma (188/320, 58.8%), cutaneous melanoma (377/988, 38.2%), colorectal adenocarcinoma (402/1519, 26.5%), and stomach adenocarcinoma (151/579, 26.1%). TLR4 was the most frequently mutated gene with eleven mutations in 10/171 (5.8%) of EAC tumors. The TLR4 mutants E439G, S570I, F703C and R787H were confirmed to have impaired reactivity to bacterial lipopolysaccharide with marked reductions in signaling by luciferase reporter assays. Overall, our findings show that TLR pathway genes are recurrently mutated in EAC, and TLR4 mutations have decreased responsiveness to bacterial lipopolysaccharide and may play a role in disease pathogenesis in a subset of patients

The oral gold compound auranofin triggers arachidonate release and cyclooxygenase metabolism in the alveolar macrophage

We examined the effect of in vitro incubation with the oral gold compound auranofin (AF) on arachidonic acid (AA) release and metabolism by rat alveolar macrophages (AMs). AF stimulated dose- and time-dependent release of 14C-AA from prelabeled AMs, which reached 4.7 +/- 0.3% (mean +/- SEM) of incorporated radioactivity at 10 [mu]g/ml for 90 min, as compared to 0.5 +/- 0.1% release following control incubation for 90 min (). Similar dose- and time-dependent synthesis of thromboxane (Tx) A2 (measured as TxB2) and prostaglandin (PG) E2 was demonstrated by radioimmunoassay of medium from unlabeled cultures, reaching 18-fold and 9-fold, respectively, of the control values at 10 [mu]g/ml AF for 90 min (p2 and PGE2 synthesis was inhibited by indomethacin as well as by pretreatment with methylprednisolone. No nicrease in the synthesis of immunoreactive leukotrienes (LT) B4 of C4 was noted at any dose or time of AF. High performance liquid chromatographic separation of 14C-eicosanoids synthesized by prelabeled AMs confirmed that AF induced the release of free AA and its metabolism to cyclooxygenase, but not 5-lipoxygenase, metabolites. The ability of AF to trigger macrophage AA metabolism may be relevant to the exacerbation of certain inflammatory processes which sometimes accompany gold therapy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27052/1/0000042.pd

Multicentre cohort study to define and validate pathological assessment of response to neoadjuvant therapy in oesophagogastric adenocarcinoma

BACKGROUND: This multicentre cohort study sought to define a robust pathological indicator of clinically meaningful response to neoadjuvant chemotherapy in oesophageal adenocarcinoma. METHODS: A questionnaire was distributed to 11 UK upper gastrointestinal cancer centres to determine the use of assessment of response to neoadjuvant chemotherapy. Records of consecutive patients undergoing oesophagogastric resection at seven centres between January 2000 and December 2013 were reviewed. Pathological response to neoadjuvant chemotherapy was assessed using the Mandard Tumour Regression Grade (TRG) and lymph node downstaging. RESULTS: TRG (8 of 11 centres) was the most widely used system to assess response to neoadjuvant chemotherapy, but there was discordance on how it was used in practice. Of 1392 patients, 1293 had TRG assessment; data were available for clinical and pathological nodal status (cN and pN) in 981 patients, and TRG, cN and pN in 885. There was a significant difference in survival between responders (TRG 1–2; median overall survival (OS) not reached) and non-responders (TRG 3–5; median OS 2·22 (95 per cent c.i. 1·94 to 2·51) years; P < 0·001); the hazard ratio was 2·46 (95 per cent c.i. 1·22 to 4·95; P = 0·012). Among local non-responders, the presence of lymph node downstaging was associated with significantly improved OS compared with that of patients without lymph node downstaging (median OS not reached versus 1·92 (1·68 to 2·16) years; P < 0·001). CONCLUSION: A clinically meaningful local response to neoadjuvant chemotherapy was restricted to the small minority of patients (14·8 per cent) with TRG 1–2. Among local non-responders, a subset of patients (21·3 per cent) derived benefit from neoadjuvant chemotherapy by lymph node downstaging and their survival mirrored that of local responders

Patient-specific cancer genes contribute to recurrently perturbed pathways and establish therapeutic vulnerabilities in esophageal adenocarcinoma

Abstract: The identification of cancer-promoting genetic alterations is challenging particularly in highly unstable and heterogeneous cancers, such as esophageal adenocarcinoma (EAC). Here we describe a machine learning algorithm to identify cancer genes in individual patients considering all types of damaging alterations simultaneously. Analysing 261 EACs from the OCCAMS Consortium, we discover helper genes that, alongside well-known drivers, promote cancer. We confirm the robustness of our approach in 107 additional EACs. Unlike recurrent alterations of known drivers, these cancer helper genes are rare or patient-specific. However, they converge towards perturbations of well-known cancer processes. Recurrence of the same process perturbations, rather than individual genes, divides EACs into six clusters differing in their molecular and clinical features. Experimentally mimicking the alterations of predicted helper genes in cancer and pre-cancer cells validates their contribution to disease progression, while reverting their alterations reveals EAC acquired dependencies that can be exploited in therapy

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

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.This study was partly funded by a project grant from Cancer Research UK. R.C.F. is funded by an NIHR Professorship and receives core funding from the Medical Research Council and infrastructure support from the Biomedical Research Centre and the Experimental Cancer Medicine Centre. We acknowledge the support of The University of Cambridge, Cancer Research UK (C14303/A17197) and Hutchison Whampoa Limited

A non-endoscopic sampling device to sample the oesophageal microbiota: a case-control study

$\textbf{Background}$ The strongest risk factor for oesophageal adenocarcinoma (OAC) is reflux disease, and the rising incidence coincides with the eradication of $\textit{Helicobacter pylori}$, both of which may alter the oesophageal microbiota. We aimed to profile the microbiota at different stages of Barrett’s carcinogenesis and investigate the Cytosponge as a minimally invasive tool for sampling the oesophageal microbiota.$\textbf{Methods}$ 16S rRNA gene amplicon sequencing was performed on 210 oesophageal samples from 86 patients representing the Barrett’s progression sequence (normal squamous controls, non-dysplastic and dysplastic Barrett’s oesophagus, and OAC), relevant negative controls and replicates on the Illumina MiSeq platform. Three different oesophageal sampling methods were compared for microbial DNA yield (qPCR), diversity and community composition: fresh frozen tissue, fresh frozen endoscopic brushings and the Cytosponge device. $\textbf{Findings}$ There was decreased microbial diversity in OAC tissue compared to normal control patients as measured by the observed OTU richness, Chao estimated total richness and Shannon diversity index (all p<0·01). $\textit{Lactobacillus fermentum}$ was enriched in OAC (p=0·028), and lactic acid bacteria dominated the microenvironment in 7 (47%) of 15 OAC cases. Comparison of oesophageal sampling methods showed that the Cytosponge yielded more than ten-fold higher quantities of microbial DNA in comparison to endoscopic brushes (p<0·001) or biopsies (p<0·0001) using qPCR. The Cytosponge samples contained the majority of taxa detected in biopsy and brush samples, but were enriched for genera from the oral cavity and stomach, including $\textit{Fusobacterium, Megasphaera, Campylobacter, Capnocytophaga and Dialister}$. The Cytosponge detected decreased microbial diversity in patients with high grade dysplasia in comparison to controls as measured by the observed OTU richness, Chao estimated total richness and Shannon diversity index (all p<0·05). $\textbf{Interpretation}$ Alterations in microbial communities occur in the lower oesophagus in Barrett’s carcinogenesis, which can be detected at the pre-invasive stage of high grade dysplasia using the novel Cytosponge.Cancer Research UK, National Institute for Health Research, Medical Research Council, and Wellcome Trust.This is the author accepted manuscript. The final version is available from Elsevier via https://doi.org/10.1016/S2468-1253(16)30086-