Esophageal Adenocarcinoma Samples Research Articles

Glucose transporter 1 expression as a marker of prognosis in oesophageal adenocarcinoma.

BackgroundThe current TNM staging system for oesophageal adenocarcinoma (OAC) has limited ability to stratify patients and inform clinical management following neo-adjuvant chemotherapy and surgery.ResultsFunctional genomic analysis of the gene expression data using Gene Set Enrichment Analysis (GSEA) identified GLUT1 as putative prognostic marker in OAC.In the discovery cohort GLUT1 positivity was observed in 114 patients (80.9%) and was associated with poor overall survival (HR 2.08, 95% CI 1.1-3.94; p=0.024) following multivariate analysis. A prognostic model incorporating GLUT1, CRM and nodal status stratified patients into good, intermediate and poor prognosis groups (p< 0.001) with a median overall survival of 16.6 months in the poorest group.In the validation set 182 patients (69.5%) were GLUT1 positive and the prognostic model separated patients treated with neo-adjuvant chemotherapy and surgery (p<0.001) and surgery alone (p<0.001) into three prognostic groups.Patients and MethodsTranscriptional profiling of 60 formalin fixed paraffin-embedded (FFPE) biopsies was performed. GLUT1 immunohistochemical staining was assessed in a discovery cohort of 141 FFPE OAC samples treated with neo-adjuvant chemotherapy and surgery at the Northern Ireland Cancer Centre from 2004-2012. Validation was performed in 262 oesophageal adenocarcinomas collected at four OCCAMS consortium centres. The relationship between GLUT1 staining, T stage, N stage, lymphovascular invasion and circumferential resection margin (CRM) status was assessed and a prognostic model developed using Cox Proportional Hazards.ConclusionsGLUT1 staining combined with CRM and nodal status identifies a poor prognosis sub-group of OAC patients and is a novel prognostic marker following potentially curative surgical resection.

Clinicopathological and functional implications of the inhibitor of apoptosis proteins survivin and XIAP in esophageal cancer.

Based on their overexpression and important roles in progression and therapy-resistance in malignant diseases, the inhibitor of apoptosis protein family (IAP) members, survivin and X-linked inhibitor of apoptosis protein (XIAP), represent attractive candidates for targeted therapy. The present study investigated the prognostic and biological relevance of survivin and XIAP in esophageal squamous-cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Survivin and XIAP expression was analyzed by immunohistochemistry using tissue microarrays containing 120 ESCC and 90 EAC samples as well as the corresponding non-neoplastic esophageal mucosa samples. IAP expression levels were then correlated to clinicopathological parameters and overall survival to identify any associations. In addition, esophageal cancer cell lines were treated with the survivin inhibitor YM155, and the XIAP inhibitors Birinapant and GDC-0152 in vitro. Survivin and XIAP expression were significantly increased in EAC and ESCC when compared with tumor-adjacent mucosa. In patients with ESCC XIAP expression was associated with female gender and advanced tumor stages, and nuclear survivin expression was associated with poor grading. High XIAP expression was identified as an independent negative prognostic marker in ESCC. By contrast, XIAP inhibitors did not affect cancer cell viability in vitro, and the small molecule survivin inhibitor YM155 significantly reduced cell viability and proliferation in esophageal cancer cell lines. Western blot analysis revealed a dose dependent decrease of survivin accompanied by an increased poly (adenosine diphosphate-ribose) polymerase cleavage following YM155 treatment. These findings underline the potential role of survivin and XIAP in the oncogenesis of esophageal cancer and provide a rationale for future clinical studies investigating the therapeutic efficacy of IAP directed therapies in patients with esophageal cancer.

LKB1 inactivation occurs in a subset of esophageal adenocarcinomas and is sufficient to drive tumor cell proliferation

BackgroundThe incidence of esophageal adenocarcinoma (EAC) has increased over the last several decades. Apart from mutations in TP53 gene, there are little data on genetic drivers of EAC. Liver kinase B1 (LKB1) has emerged as a multifunctional tumor suppressor regulating cell growth, differentiation, and metabolism. Somatic inactivation of LKB1 has been described in several tumor types; however, whether LKB1 inactivation has a role in EAC is unknown. Here we analyzed patient tumors to assess the prevalence of LKB1 loss in EAC. MethodsChromosomal deletion and expression of LKB1 in EAC were investigated using publicly available genomic data. Protein expression was assessed by immunohistochemistry (IHC) analysis for LKB1 in a tissue microarray (TMA) containing esophageal tumor specimens, including EAC. LKB1 was suppressed in EAC cells to determine the effects on cell growth in vitro. ResultsAnalysis of EAC data in The Cancer Genome Atlas dataset revealed significant deletion of chromosome 19p13.3, containing the LKB1 gene locus. Single copy loss (shallow deletion) of LKB1 was present in 58% of EAC samples. Expression of LKB1 was significantly lower in EAC tumors compared with normal esophagus. IHC analysis showed reduced LKB1 protein expression in EAC. Suppression of LKB1 was sufficient to enhance EAC cell growth in vitro. ConclusionsOur data suggest that inactivation of LKB1 frequently occurs in EAC. Based on the reported oncogenic effects of LKB1 inactivation, our data indicate that LKB1 loss may play a significant role in EAC tumorigenesis, and point to the need for future studies.

Abstract 2746: Detection of tumor-specific mutations in plasma DNA: A potential esophageal adenocarcinoma biomarker

Abstract BACKGROUND: Recent studies have shown that tumor-specific DNA from multiple types of tumors can be detected circulating in plasma and this has raised the possibility of “liquid biopsies” using mutated tumor DNA as a potential diagnostic and prognostic biomarker. Detection of mutations with allele frequencies below 0.1% remains challenging however given that circulating cell-free DNA is highly degraded and in low abundance. Detection of multiple different mutations in the same sample presents an additional challenge particularly when the mutation panel may change from patient to patient. We have developed a novel approach, called SimSen-Seq, to introduce molecular barcodes into sequencing libraries with DNA inputs as low as 5ng. Barcodes enable differentiation of true mutants from background noise introduced by Taq polymerase errors and permits detection of variant alleles with frequencies below 0.1%. The barcodes are protected from mis-priming using a hairpin structure which permits a high degree of multiplexing and flexibility for detection of multiple mutations from one plasma sample. We are using this technology to test the utility of liquid biopsy as a biomarker for esophageal adenocarcinoma (EAC) diagnosis and disease monitoring. METHODS: Blood samples were obtained at a single time point from patients with various stages of EAC and longitudinal blood samples were also collected from patients undergoing neoadjuvant therapy followed by surgery. Tumor samples were obtained from biopsy or resection specimens. Tumor DNA was sequenced using a targeted EAC panel to identify mutations in each tumor. SimSen-Seq assays were designed to identify these mutations in plasma, and hairpin barcodes were attached. Sequencing libraries were generated from circulating DNA, sequenced and analyzed using the barcodes to reduce background noise. RESULTS: Mutations were identified in tumor samples from 37 patients. To date, 29 patients have had plasma analyzed; 5 stage I, 6 stage II, 13 stage III, and 5 stage IV. Of these 29, the same mutations have been identified in 15 plasma DNA samples (20% stage I, 50% stage II, 54% stage III, 80% stage IV. Six patients demonstrated multiple mutant alleles in plasma DNA. One patient with detectable pre-treatment ctDNA underwent serial blood draws during their treatment course, and post-operative detection of tumor-specific markers preceded physical evidence of disease recurrence. DISCUSSION: SimSen-Seq shows promise as a novel ultra-sensitive, highly multiplexed sequencing method for identifying rare circulating mutations. Possible applications include prognostication in early stage patients and rapid monitoring of therapeutic response and recurrence. Further work is to evaluate this is ongoing. Citation Format: Matthew Egyud, Jennifer Jackson, Emiko Yamada, Anders Ståhlberg, Paul Krzyzanowski, Virginia Litle, Lincoln Stein, Tony Godfrey. Detection of tumor-specific mutations in plasma DNA: A potential esophageal adenocarcinoma biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2746. doi:10.1158/1538-7445.AM2017-2746

Abstract 4375: Complex interactions between genomic and epigenomic alterations in esophageal adenocarcinomas

Abstract Background: The incidence of Barrett’s-related esophageal adenocarcinoma (EAC) is rapidly rising in the USA and the Western countries. However, the interaction between genomic and epigenomic alterations in regulating gene expression networks in EAC is poorly understood. Results and Discussion: We carried out an integrative analytical approach to identify DNA copy number, promoter DNA methylation and gene expression in a panel of esophageal adenocarcinomas, using chip-based technologies. Our analysis displayed significant differences between EAC and normal samples in DNA copy numbers, gene expression, and DNA methylation profiles. Our results identified remarkable incidence of DNA copy number (CN) alterations showing that 90% of probes had a copy number aberration (gain or loss) in at least one tumor, suggesting a vast chromosomal instability in EAC. Integrative analysis indicated that altered expression of 1755 genes was associated with changes in CN or methylation. We found that expression alterations in 84 genes (56 overexpressed and 28 down regulated) were associated with changes in both CN and methylation. These data suggest a strong interaction between genetic and epigenetic events to modulate gene expression in EAC. Of note, we detected a prominent K-RAS signature in EAC. Network analysis predicted activation of several important transcription factor networks in EAC, including β-catenin, MYB, TWIST1, SOX7, GATA3 and GATA6. 56 genes were overexpressed in EAC with simultaneous promoter hypomethylation and gene amplification, including CDH17 and GATA6. Notably, several genes associated with inflammation such as COX2, IL8 and IL23R were overexpressed and hypomethylated in EAC, a reaffirmation of the underlying inflammatory process in Barrett’s tumorigenesis and a suggestion of an epigenetic regulation of these genes. Conclusions: In summary, our integrative analysis of genetic and epigenetic alterations in EAC demonstrates a complex interaction between molecular mechanisms in EAC and provides several novel insights for future studies. Citation Format: Dunfa Peng, Yan Guo, Heidi Chen, Shilin Zhao, Kay Washington, Tianling Hu, Yu Shyr, Wael El-Rafai. Complex interactions between genomic and epigenomic alterations in esophageal adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4375. doi:10.1158/1538-7445.AM2017-4375

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 3815: Cyclin-dependent kinase 9: a potential therapeutic target for esophageal adenocarcinoma

Abstract Objective: Major limitation of successful implementation of targeted therapy in esophageal adenocarcinoma (EAC) is low frequency of target specific biomarker alteration and intratumoral heterogeneity. Cyclin dependent kinase 9 (CDK9) is a ubiquitous serine threonine kinase whoseinhibition has been used as a therapeutic option in other solid tumors. However, critical in vitro and in vivo information about the efficacy and mechanism of CDK9 inhibition in EAC is lacking and needs to be generated before a clinical trial is proposed. Design: We assessed the CDK9 protein expression in EAC cell lines and in patient tissue samples of Barrett's esophagus and EAC. Subsequently, we studied effects of genetic downregulation (shCDK9) in SKGT4 and chemical inhibition of CDK9 by CDK9 inhibitor II, a specific, potent competitive inhibitor of CDK9 (Santa Cruz Biotechnology, CA) on proliferation, apoptosis, and cell cycle of SKGT4, FLO-1 and OE33. In vivo effects of shCDK9 and CDK9 inhibitor II was analyzed in xenografts experiments performed on nude mice after injection of FLO-1 or shCDK9-SKGT 4 and control cells in right flank or abdomen. We also assessed the effects of shCDK9 and CDK9 inhibitor II on expression of MCL-1 and phosphorylation of RNA polymerase II in EAC cells. Results: Higher expression of CDK9 was observed in all EAC cell lines and in tumor samples of patients with EAC as compared to normal squamous epithelium and Barrett's Esophagus. shCDK9 in SKGT4 cells induced significant reduction in proliferation, increase in apoptosis and G1 arrest as compared to control cells in vitro. In vivo experiment with SKGT4-shCDK9 demonstrated no tumor growth in 4 of 10 mice and tumor volume was significantly smaller in SKGT4-shCDK9 as compared to control SKGT4 cells (72.89 ± 12.88 mm3 v.270 ± 64.07 mm3, p&amp;lt; 0.01) in other mice. Treatment with CDK9 inhibitor II demonstrated dose dependent reduction in proliferation, increased apoptosis and G1 arrest in all three cell lines in vitro and more than 50% reduction in tumor volume in xenografts as compared to control. CDK9 inhibitor II reduced RNA Pol II phosphorylation and expression of MCL-l and up-regulation of MCL-1 expression induced resistance to CDK9 inhibitor II in all three EAC cell lines.. Conclusions: CDK9 inhibition has strong anti-EAC properties in vitro and in vivo. These properties along with strong diffuse expression of CDK9 in human EAC support further exploration of CDK9 as a potential therapeutic target for EAC. Citation Format: Zhimin Tong, Devkumar Chatterjee, Defeng Deng, Omkara Veeranki, Alicia Mejia, Jaime Rodriguez, Jaffer Ajani, Wayne Hofstetter, Steven Lin, Sushovan Guha, Scott Kopetz, Sunil Krishnan, Dipen Maru. Cyclin-dependent kinase 9: a potential therapeutic target for esophageal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3815.

Elevated expression of the IGF2 mRNA binding protein 2 (IGF2BP2/IMP2) is linked to short survival and metastasis in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) represents the sixth leading cause of cancer-related deaths and develops in Barret's esophagus affected tissues. The IGF2 mRNA binding protein IMP2/IGF2BP2/p62 was originally identified as an autoantigen in hepatocellular carcinoma. Aim of this study was to investigate the expression and prognostic role of IMP2 in EAC. Human EAC and Barret's esophagus tissue showed overexpression of IMP2, particularly in tumors of increased size and in metastatic tissues. Molecular classification based on published gene signatures of esophageal cancer revealed a specific subtype, in which the expression of IMP2 is high. According to GO and KEGG pathway analyses, genes showing highly correlated expression with IMP2 are associated with growth, proliferation, metabolism, inflammation, and cancerous processes. Clustering of EAC samples according to published survival marker genes strongly suggests that IMP2 overexpressing samples show poor survival. Finally, IMP2 expression correlated with short survival in patients with EAC or esophageal squamous carcinoma. Our data indicate that IMP2 might be a useful prognostic marker for Barret's esophagus and EAC.

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.

HMGA2 overexpression plays a critical role in the progression of esophageal squamous carcinoma.

Esophageal Squamous Cell Carcinoma (ESCC) is the most common esophageal tumor worldwide. However, there is still a lack of deeper knowledge about biological alterations involved in ESCC development. High Mobility Group A (HMGA) protein family has been related with poor outcome and malignant cell transformation in several tumor types. In this way, the aim of this study was to analyze the expression of HMGA1 and HMGA2 expression in ESCC and their role in crucial cellular features. We evaluated HMGA1 and HMGA2 mRNA expression in 52 paired ESCC and normal surrounding tissue samples by qRT-PCR. Here, we show that HMGA2, but not HMGA1, is overexpressed in ESCC samples. This result was further confirmed by the immunohistochemical analysis. Indeed, accordingly to mRNA expression data, HMGA2, but not HMGA1, was overexpressed in approximately 90% of ESCC samples, while it was barely expressed in the respective control. Conversely, HMGA1, but not HMGA2, was overexpressed in esophageal adenocarcinoma samples. Interestingly, HMGA2 abrogation attenuated the malignant phenotype of two ESCC cell lines, suggesting that HMGA2 overexpression is involved in ESCC progression.

The Barrett-associated variants at GDF7 and TBX5 also increase esophageal adenocarcinoma risk.

Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) represent two stages within the esophagitis‐metaplasia‐dysplasia‐adenocarcinoma sequence. Previously genetic risk factors have been identified that confer risk to BE and EAC development. However, to which extent the genetic variants confer risk to different stages of the BE/EAC sequence remains mainly unknown. In this study we analyzed three most recently identified BE variants at the genes GDF7 (rs3072), TBX5 (rs2701108), and ALDH1A2 (rs3784262) separately in BE and EAC samples in order to determine their risk effects during BE/EAC sequence. Our data show that rs3072 at GDF7 and rs2701108 at TBX5 are also associated with EAC and conclude that both loci confer disease risk also at later stages of the BE/EAC sequence. In contrast, rs3784262 at ALDH1A2 was highly significantly associated with BE, but showed no association with EAC. Our data do not provide evidence that the ALDH1A2 locus confers equal risk in early and late stages of BE/EAC sequence.

Biobanking of fresh-frozen endoscopic biopsy specimens from esophageal adenocarcinoma.

The process of preparing endoscopic esophageal adenocarcinoma samples for next-generation DNA/RNA sequencing is poorly described. Therefore, we assessed the feasibility and pitfalls of preparing esophageal adenocarcinoma endoscopic biopsies toward DNA/RNA samples suitable for next-generation sequencing. In this prospective study, four tumor biopsy samples were collected from consecutive esophageal cancer patients during esophagogastroduodenoscopy and fresh-frozen in liquid nitrogen. DNA and RNA were isolated from samples with a tumor percentage of at least 50%. For next-generation sequencing, double-stranded DNA (dsDNA) is required and high-quality RNA preferred. The quantity dsDNA and RNA quantity and quality were assessed with the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Biopsy samples of 69 consecutive patients with esophageal adenocarcinoma were included. In five patients (7%), the tumor percentage was less than 50% in all four biopsies. Using a protocol allowing simultaneous DNA and RNA isolation, the median dsDNA yield was 2.4 μg (range 0.1-12.0 μg) and the median RNA yield was 0.5 μg (range 0.01-2.05 μg). The median RNA integrity number of samples that were fresh-frozen within 30 minutes after sampling was 6.7 (range 4.2-8.9) compared with 2.5 (1.8-4.5) for samples that were fresh-frozen after 2 hours. The results from this study show that obtaining dsDNA and RNA for next-generation sequencing from endoscopic esophageal adenocarcinoma samples is feasible. Tumor percentage and dsDNA/RNA yield and quality emphasize the need for sampling multiple biopsies and minimizing the delay before fresh-freezing.

Copy number alterations detected by whole-exome and whole-genome sequencing of esophageal adenocarcinoma.

BackgroundEsophageal adenocarcinoma (EA) is among the leading causes of cancer mortality, especially in developed countries. A high level of somatic copy number alterations (CNAs) accumulates over the decades in the progression from Barrett’s esophagus, the precursor lesion, to EA. Accurate identification of somatic CNAs is essential to understand cancer development. Many studies have been conducted for the detection of CNA in EA using microarrays. Next-generation sequencing (NGS) technologies are believed to have advantages in sensitivity and accuracy to detect CNA, yet no NGS-based CNA detection in EA has been reported.ResultsIn this study, we analyzed whole-exome (WES) and whole-genome sequencing (WGS) data for detecting CNA from a published large-scale genomic study of EA. Two specific comparisons were conducted. First, the recurrent CNAs based on WGS and WES data from 145 EA samples were compared to those found in five previous microarray-based studies. We found that the majority of the previously identified regions were also detected in this study. Interestingly, some novel amplifications and deletions were discovered using the NGS data. In particular, SKI and PRKCZ detected in a deletion region are involved in transforming growth factor-β pathway, suggesting the potential utility of novel biomarkers for EA. Second, we compared CNAs detected in WGS and WES data from the same 15 EA samples. No large-scale CNA was identified statistically more frequently by WES or WGS, while more focal-scale CNAs were detected by WGS than by WES.ConclusionsOur results suggest that NGS can replace microarrays to detect CNA in EA. WGS is superior to WES in that it can offer finer resolution for the detection, though if the interest is on recurrent CNAs, WES can be preferable to WGS for its cost-effectiveness.Electronic supplementary materialThe online version of this article (doi:10.1186/s40246-015-0044-0) contains supplementary material, which is available to authorized users.

Analysis of Dysplasia in Patients With Barrett’s Esophagus Based on Expression Pattern of 90 Genes

Diagnoses of dysplasia, based on histologic analyses, dictate management decisions for patients with Barrett's esophagus (BE). However, there is much intra- and inter-observer variation in identification of dysplasia-particularly low-grade dysplasia. We aimed to identify a biomarker that could be used to assign patients with low-grade dysplasia to a low- or high-risk group. We performed a stringent histologic assessment of 150 frozen esophageal tissues samples collected from 4 centers in the United Kingdom (from 2000 through 2006). The following samples with homogeneous diagnoses were selected for gene expression profiling: 28 from patients with nondysplastic BE, 10 with low-grade dysplasia, 13with high-grade dysplasia (HGD), and 8 from patients with esophageal adenocarcinoma. A leave-one-out cross-validation analysis was used identify a gene expression signature associated with HGD vs nondysplastic BE. Functional pathways associated with gene signature sets were identified using the MetaCore analysis. Gene expression signature sets were validated using gene expression data on BE and esophageal adenocarcinoma accessed through National Center for Biotechnology Information Gene Expression Omnibus, as well as a separate set of samples (n=169) collected from patients who underwent endoscopy in the United Kingdom or the Netherlands and analyzed histologically. We identified an expression pattern of 90 genes that could separate nondysplastic BE tissues from those with HGD (P < .0001). Genes in a pathway regulated by retinoic acid-regulated nuclear protein made the largest contribution to this gene set (P < .0001); the transcription factor MYC regulated at least 30% of genes within the signature (P < .0001). In the National Center for Biotechnology Information Gene Expression Omnibus validation set, the signature separated nondysplastic BE samples from esophageal adenocarcinoma samples (P=.0012). In the UK and Netherlands validation cohort, the signature identified dysplastic tissues with an area under the curve value of 0.87(95% confidence interval: 0.82-0.93). Of samples with low-grade dysplasia (LGD), 64%were considered high risk according to the 90-gene signature; these patients had a higher rate of disease progression than those with a signature categorized as low risk (P=.047). We identified an expression pattern of 90 genes in esophageal tissues of patients with BE that was associated with low- or high-risk for disease progression. This pattern might be used in combination with histologic analysis of biopsy samples to stratify patients for treatment. It would be most beneficial for analysis of patients without definitive evidence of HGD but for whom early endoscopic intervention is warranted.

Abstract 4006: MiRNA profiling in esophageal precancerous for malignancy progression risk assessment

Abstract Background: Barrett's esophagus (BE) is a metaplastic disease with risk to develop esophageal adenocarcinoma (EAC). Current screening method for BE patients (endoscopy and biopsy) is discomfort, expensive and relatively risky for patients. Moreover, the procedure is complicated by subjective histopathology examination and only 2% of BE patients annually progress to adenocarcinoma. The aim of our study was to identify miRNA profiles in order to predict cancer progression in high-risk BE patients. Methods and patients: FFPE samples were obtained from 113 patients (35 EAC, 21 low-grade dysplasia of esophagus, 33 BE and 24 healthy control). There were 74 males and 39 females; mean age was 55.9 years, range 15 - 94 years. Total RNA was purified from FFPE biopsy samples using proteinase K treatment followed by RNeasy kit (Qiagen). Microarray analysis was performed using the GeneChip miRNA 3.0 Array (Affymetrix). The data were analyzed using “R” software and the Bioconductor package. Results: In total, 113 GeneChip miRNA 3.0 Arrays were performed in 113 patients. Expression profiles of 1733 human miRNAs were analyzed. In supervised clustering analysis we found 8 differentially expressed miRNAs (p&amp;lt;0.001 or logFC 2) in BE patients compared to EAC patients and 10 differentially expressed miRNAs (p&amp;lt;0.001 or logFC 2) in LGD patients compared to EAC patients. One of the dominant findings was decreased expression of miR-223 in EAC and LGD samples, which is known to function as a tumor suppressor blocking E2F1. Using LOOCV classifier with 13 miRNAs signature we were able to correctly classify 86 from 113 samples (AUC = 0.840). Prediction model fails mostly in LGD prediction (18/21), probably due to incorrect pathological classification. Conclusion: We have identified miRNA profiles and signature allowing for better diagnostics of cancer progression in BE and LGD patients, which, however, will require further validation. Acknowledgment: This work was financially supported by IGA UP LF 2014_019, NPU LO1304, CZ.1.05/2.1.00/01.0030 and CZ.1.07/2.3.00/30.0004. Citation Format: Josef Srovnal, Ondrej Slaby, Jiri Ehrmann, Jan Gregar, Lenka Radova, Katerina Staffova, Marian Hajduch. MiRNA profiling in esophageal precancerous for malignancy progression risk assessment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4006. doi:10.1158/1538-7445.AM2015-4006

Whole-genome sequencing provides new insights into the clonal architecture of Barrett's esophagus and esophageal adenocarcinoma.

The molecular genetic relationship between esophageal adenocarcinoma (EAC) and its precursor lesion, Barrett’s esophagus, is poorly understood. Using whole-genome sequencing on 23 paired Barrett’s esophagus and EAC samples, together with one in-depth Barrett’s esophagus case-study sampled over time and space, we have provided new insights on the following aspects: i) Barrett’s esophagus is polyclonal and highly mutated even in the absence of dysplasia; ii) when cancer develops, copy number increases and heterogeneity persists such that the spectrum of mutations often shows surprisingly little overlap between EAC and adjacent Barrett’s esophagus; and iii) despite differences in specific coding mutations the mutational context suggests a common causative insult underlying these two conditions. From a clinical perspective, the histopathological assessment of dysplasia appears to be a poor reflection of the molecular disarray within the Barrett’s epithelium and a molecular Cytosponge™ technique overcomes sampling bias and has capacity to reflect the entire clonal architecture.

PWE-177 The functional relevance of toll-like receptor 4 mutations in oesophageal adenocarcinoma

Introduction Altered innate immune signalling may disrupt host-microbe homeostasis and promote an inflammatory microenvironment that favours tumourigenesis. The aim of this project was to assess whether somatic mutations in oesophageal adenocarcinoma (OAC) could interfere with Toll-like receptor (TLR) signalling, with a specific focus on TLR4. Method We interrogated the mutational profiles of TLR genes in 170 OAC samples, which had undergone whole genome sequencing as part of the oesophageal ICGC study. We performed site-directed mutagenesis and created 13 plasmids expressing mutant TLR4 with a CMV promoter (12 single nucleotide variants (SNVs), and one plasmid combined two SNVs). Luciferase reporter gene assays were performed in HEK293 cells stimulated with synthetic monophosphoryl lipid A (MPLA) to assess how the mutants altered TLR4 signalling and downstream activation of nuclear factor kappa B (NF-κB). Two of the TLR4 mutants were taken forward for transfection into the OAC cell line OE33, and ELISA was used to measure cytokine secretion after stimulation with MPLA. Results The TLR signalling pathway appeared to be recurrently mutated with missense mutations in 23/170 (13.5%) of tumour samples, including mutations in TLR1 (1.2%), TLR2 (0.6%), TLR4 (4.7%), TLR5 (0.6%), TLR7 (1.8%), TLR9 (1.2%), and MYD88 (1.5%). Based on our data and another recent study by Dulak et al . 1 , TLR4 mutations were the most frequent (5.4% combined data). One tumour sample contained two TLR4 mutations, E439G and F703C. Interestingly, the mutation E439G was identified both in our dataset and in the study by Dulak et al . Furthermore, in silico modelling suggested that the E439G mutation disrupts hydrogen bonds in the binding site of LPS and MD2. The use of NF-κB luciferase reporter assays demonstrated a significant decrease in ligand-dependent signalling for 8/12 of the TLR4 mutations tested in HEK293 cells. A double mutation of E439G with F703C revealed a further decrease in TLR4 signalling. Similarly, the concentration of IL-8 was lower for TLR4 mutants E439G and E439G+F703C transfected into OE33 cells stimulated with MPLA, in comparison to wild-type TLR4. Conclusion TLR4 is recurrently mutated in OAC, and the majority of these mutations showed decreased TLR4 signalling in response to ligand stimulation, both in HEK293 and OE33 cells. The biological relevance of this in relation to host-microbe homeostasis and carcinogenesis is being explored. Disclosure of interest None Declared. Reference Dulak AM, et al . Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity. Nat Genet. 2013;45(5):478–486.

Abstract 1492: Prediction of cancer progression in Barrett's esophagus patients using miRNA profiling

Abstract Background: Barrett's esophagus (BE) is a metaplastic disease with risk to develop esophageal adenocarcinoma (EAC). Current screening method for BE patients (endoscopy and biopsy) is discomfort, expensive and relatively risky for patients. Moreover, the procedure is complicated by subjective histopathological examination. Approximately only 2% of BE patients annually progress to adenocarcinoma. The aim of our study was to identify miRNA profiles in order to predict cancer progression in high-risk BE patients. Methods and patients: FFPE samples were obtained from 68 patients (10 EAC, 4 high-grade and 21 low-grade dysplasia of esophagus, and 33 BE). There were 49 males and 19 females; median age was 58 years, range 27 - 97 years. Total RNA was purified from FFPE biopsy samples using Proteinase K treatment followed by RNeasy kit (Qiagen). Microarray analysis was performed using the GeneChip miRNA 3.0 Array (Affymetrix). The data were analyzed using “R” software and the Bioconductor package. Results: In total, 68 GeneChip miRNA 3.0 Arrays were performed in 68 patients (8 poor quality arrays were excluded). Expression profiles of 1733 human miRNAs, 1658 human pre-miRNAs and 2216 human sno-RNAs and sca-RNAs were analyzed. In supervised clustering analysis we found 29 differentially expressed miRNAs, 7 pre-miRNAs and 9 sno-RNAs (p&amp;lt;0.001 or logFC &amp;lt;-2 or logFC &amp;gt;2) in EAC patients compared to BE patients. One of the dominant findings was decreased expression of miR-22 in EAC samples, which is known to function as a tumor suppressor and histone deacetylase 4 regulator. Conclusion: We have identified miRNA profiles allowing for better diagnostics of cancer progression in BE patients, which, however, will require further validation. Acknowledgment: This work was financially supported by IGA MZ CR NT13585-3/202, IGA UP LF 2013_015, CZ.1.05/2.1.00/01.0030 and CZ.1.07/2.3.00/30.0004. Citation Format: Josef Srovnal, Ondrej Slaby, Jiri Ehrmann, Jan Gregar, Lenka Radova, Michaela Sedlackova, Marian Hajduch. Prediction of cancer progression in Barrett's esophagus patients using miRNA profiling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1492. doi:10.1158/1538-7445.AM2014-1492

OC-016 The Toll-like Receptor Pathway Is Recurrently Mutated In Oesophageal Adenocarcinoma

IntroductionThe interaction between the oesophageal microbiota and the inflammatory microenvironment in Barrett’s carcinogenesis is poorly understood. One of the mechanisms by which microbiota may induce chronic inflammation is by triggering...

Sa1843 The Toll-Like Receptor Pathway Is Recurrently Mutated in Esophageal Adenocarcinoma

Introduction The interaction between the oesophageal microbiota and the inflammatory microenvironment in Barrett’s carcinogenesis is poorly understood. One of the mechanisms by which microbiota may induce chronic inflammation is by triggering Toll-like receptor (TLR) signalling and activation of nuclear factor kappa B. We aimed to utilise whole genome sequencing (WGS) data to investigate TLR mutations and expression in oesophageal adenocarcinoma (OAC), with a focus on TLR9. Methods We interrogated the mutational profiles of 66 OAC samples, with matched germline references from each case, which had undergone WGS as part of the oesophageal ICGC study. All mutations were verified using PCR and Sanger sequencing. To further explore TLR9 expression along the Barrett’s progression sequence, we performed TLR9 immunohistochemistry on tissue microarray samples including normal squamous oesophagus (N=16), duodenum (N=14), non-dysplastic Barrett’s (N=53), low-grade dysplasia (N=13), high-grade dysplasia (N=25) and OAC (N=338). Within the large cohort of OAC samples we binarised the intensity scores (0–1 and 2–3) and examined whether there were any significant differences in relation to clinicopathologic variables (TNM stage, histological grade, lymphovascular invasion, survival). Results We identified missense mutations in TLR pathway genes in 8/66 (12.1%) of OAC samples, including TLR1 (1.5%), TLR4 (3%), TLR7 (1.5%), TLR9 (3%), MYD88 (1.5%), and TRAF6 (1.5%). TLR9 protein was expressed more highly in Barrett’s and OAC than normal oesophageal squamous tissue (p Conclusion TLR pathway genes appear to be recurrently mutated in OAC, which given the mutational context and heterogeneity of disease 1 could represent significant involvement of the TLR signalling pathway in Barrett’s carcinogenesis. Reference 1 Dulak AM, et al . Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity. Nat Genet . 2013 May;45(5):478-86 Disclosure of Interest None Declared.