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

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-

Case report: recurrent metastatic breast cancer in internal mammary dissection bed discovered at the time of coronary bypass.

IntroductionMany patients who undergo coronary artery bypass surgery have a prior history of cancer and potentially chest radiation which is a known risk factor for coronary atherosclerosis. Prior radiation increases fibrosis and can make the dissection of the left internal mammary artery (LIMA) more challenging.Case reportA 72-year-old woman with a history of stage IIA pT2N0M0 left breast intraductal carcinoma treated with lumpectomy, adjuvant chemotherapy and radiation therapy 11 years prior presented to the emergency room with a non-ST elevation myocardial infarction and was taken for cardiac catheterization followed by three-vessel coronary artery bypass grafting. The LIMA was found to be encased in scar tissue and was deemed unsuitable as a conduit, and a saphenous vein graft was bypassed to the left anterior descending artery in its place. Pathologic review of the LIMA showed nests of tumor cells infiltrating within dense fibrous tissue with areas of necrosis and calcifications consistent with recurrent breast cancer. Interestingly the patients original breast cancer was positive for estrogen receptors (ER) and progesterone receptors (PR) ER and PR and negative for HER2 and she had therefore been treated with 5 years of hormonal therapy. The recurrent cancer found in the LIMA dissection bed at the time of bypass surgery was ER, PR, and HER2 negative, suggesting hormonal therapy driven clonal selection of these metastatic tumor cells.Discussion and conclusionsScarring in the LIMA dissection bed in patients with a history of cancer and prior chest radiation should be carefully evaluated for the possibility of recurrent cancer. The gross appearance of tissue can be misleading and sending a biopsy for a formal frozen section histologic evaluation should be considered if there is any question of recurrent malignancy

Case report: recurrent metastatic breast cancer in internal mammary dissection bed discovered at the time of coronary bypass.

IntroductionMany patients who undergo coronary artery bypass surgery have a prior history of cancer and potentially chest radiation which is a known risk factor for coronary atherosclerosis. Prior radiation increases fibrosis and can make the dissection of the left internal mammary artery (LIMA) more challenging.Case reportA 72-year-old woman with a history of stage IIA pT2N0M0 left breast intraductal carcinoma treated with lumpectomy, adjuvant chemotherapy and radiation therapy 11 years prior presented to the emergency room with a non-ST elevation myocardial infarction and was taken for cardiac catheterization followed by three-vessel coronary artery bypass grafting. The LIMA was found to be encased in scar tissue and was deemed unsuitable as a conduit, and a saphenous vein graft was bypassed to the left anterior descending artery in its place. Pathologic review of the LIMA showed nests of tumor cells infiltrating within dense fibrous tissue with areas of necrosis and calcifications consistent with recurrent breast cancer. Interestingly the patients original breast cancer was positive for estrogen receptors (ER) and progesterone receptors (PR) ER and PR and negative for HER2 and she had therefore been treated with 5 years of hormonal therapy. The recurrent cancer found in the LIMA dissection bed at the time of bypass surgery was ER, PR, and HER2 negative, suggesting hormonal therapy driven clonal selection of these metastatic tumor cells.Discussion and conclusionsScarring in the LIMA dissection bed in patients with a history of cancer and prior chest radiation should be carefully evaluated for the possibility of recurrent cancer. The gross appearance of tissue can be misleading and sending a biopsy for a formal frozen section histologic evaluation should be considered if there is any question of recurrent malignancy

TLR pathway mutations in EAC.

<p>The EAC samples with TLR pathway mutations in the (A) ICGC cohort (n = 171 patients) and (B) TCGA cohort (n = 149 patients), with gene names along the vertical axis and sample names along the horizontal axis. The mutation status for known driver mutations in EAC (<i>TP53</i>, <i>CDKN2A</i>, <i>SMAD4</i>, <i>PIK3CA</i>) is presented for each sample with TLR pathway mutations in the ICGC and TCGA cohorts. Recurring oncogenic amplifications (<i>ERBB2</i>, <i>EGFR</i>, <i>CCND1</i>) and deletions (<i>CDKN2A/B</i>) that are known drivers in EAC are presented for the ICGC cohort. LY96 is the gene encoding MD2 protein. (C) Total TLR pathway mutation frequency from the combined ICGC and TCGA cohorts (n = 320), with gene names along the vertical axis and percent of mutated samples along the horizontal axis. (D) Schematic of the TLR signaling pathway summarizing the somatic mutations from the combined ICGC and TCGA cohorts, with mutated pathway components highlighted in color and large bold font. Grey pathway components have no TLR pathway mutations in either cohort.</p

Clinicopathologic characteristics grouped by TLR pathway mutation status.

<p>Clinicopathologic characteristics grouped by TLR pathway mutation status.</p

Distribution of TLR pathway mutations in EAC and other cancers.

<p>(A) TLR pathway mutations in different cancer types from the COSMIC database, with cancer types along the vertical axis and percent of mutated samples along the horizontal axis (abbreviations provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006808#pgen.1006808.s003" target="_blank">S3 Table</a>). <i>TLR4</i> mutations are indicated in dark blue. (B) Schematic of TLR4 protein with mutations from the ICGC (green circles) and TCGA (purple circles) EAC cohorts. Both EAC cohorts identified missense mutations at amino acid position E439 and F487. (C) Schematic of TLR4 protein with mutations from different cancer types in the COSMIC database. (D) Three lanes show dimer contacts (to other TLR4 chain), MD2, and LPS (PDB ID: 4G8A [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006808#pgen.1006808.ref022" target="_blank">22</a>]). The fourth lane was calculated from a homology model of TLR4 TIR domain dimer (based on PDB ID: 2J67 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006808#pgen.1006808.ref023" target="_blank">23</a>]). Contacting TLR4 residues with atoms less than 0.5 Å apart at surfaces were determined using Chimera findclash function. Numbers of contacts are plotted in 10 residue sections of increasingly dark red tones (maximum 8 is black).</p

Functional analysis of <i>TLR4</i> mutations.

<p>(A) NF-κB response to TLR4 ligands in HEK293 cells transfected with <i>TLR4</i> mutants <i>versus</i> wild-type <i>TLR4</i> and stimulated with 100 ng/ml synthetic Lipid A and 10ng/ml LPS. The y-axis is normalized luminescence, calculated by dividing NF-κB firefly luciferase by the housekeeper renilla luciferase. Results are the average of three experiments with all conditions performed in triplicate. Error bars are standard deviation. *p<0.05, **p<0.01, ***p<0.001. (B) TLR4 protein expression 48 hours after transfection for NF-κB luciferase reporter assays. Representative Western blot shows protein expression for nine <i>TLR4</i> mutants, the double mutant (E439G + F703C), wild-type <i>TLR4</i> and empty vector. Fully glycosylated TLR4 is the upper band (120 kDa), and deglycosylated TLR4 is the lower band (110 kDa). (C) Immunofluorescence staining FLAG-AlexaFluor 488 antibody (green) in HEK293 cells transfected with wild-type TLR4-FLAG shows cytoplasmic distribution, in comparison to empty vector. Mutant E439G and R787H did not show any difference in localization of TLR4 protein. DAPI (blue) and Phalloidin (red) were used to visualize the cell nucleus and cytoskeleton, respectively. (D) Fold-change secretion of IL-8 for the EAC cell lines OE33 and JH-EsoAd1 following transfection of <i>TLR4</i> mutants E439G, R787H, E439G+F703C and wild type <i>TLR4</i>. Cells were stimulated with synthetic MPLA and LPS for 24 hours prior to ELISA. Data were normalized by dividing the concentration of IL-8 by the unstimulated (nil) value for each transfection condition. Data are an average of four independent experiments performed in duplicate. Error bars are standard deviation. *p<0.05, **p<0.01,</p

Concerted epithelial and stromal changes during progression of Barrett’s Esophagus to invasive adenocarcinoma exposed by multi-scale, multi-omics analysis

Esophageal adenocarcinoma arises from Barrett's esophagus, a precancerous metaplastic replacement of squamous by columnar epithelium in response to chronic inflammation. Multi-omics profiling, integrating single-cell transcriptomics, extracellular matrix proteomics, tissue-mechanics and spatial proteomics of 64 samples from 12 patients' paths of progression from squamous epithelium through metaplasia, dysplasia to adenocarcinoma, revealed shared and patient-specific progression characteristics. The classic metaplastic replacement of epithelial cells was paralleled by metaplastic changes in stromal cells, ECM and tissue stiffness. Strikingly, this change in tissue state at metaplasia was already accompanied by appearance of fibroblasts with characteristics of carcinoma-associated fibroblasts and of an NK cell-associated immunosuppressive microenvironment. Thus, Barrett's esophagus progresses as a coordinated multi-component system, supporting treatment paradigms that go beyond targeting cancerous cells to incorporating stromal reprogramming