Nicholas Shannon

Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis

Cancer genome sequencing studies have identified numerous driver genes, but the relative timing of mutations in carcinogenesis remains unclear. The gradual progression from premalignant Barretts esophagus to esophageal adenocarcinoma (EAC) provides an ideal model to study the ordering of somatic mutations. We identified recurrently mutated genes and assessed clonal structure using whole-genome sequencing and amplicon resequencing of 112 EACs. We next screened a cohort of 109 biopsies from 2 key transition points in the development of malignancy: benign metaplastic never-dysplastic Barretts esophagus (NDBE; n = 66) and high-grade dysplasia (HGD; n = 43). Unexpectedly, the majority of recurrently mutated genes in EAC were also mutated in NDBE. Only TP53 and SMAD4 mutations occurred in a stage-specific manner, confined to HGD and EAC, respectively. Finally, we applied this knowledge to identify high-risk Barretts esophagus in a new non-endoscopic test. In conclusion, mutations in EAC driver genes generally occur exceptionally early in disease development with profound implications for diagnostic and therapeutic strategies.

Stromal genes discriminate preinvasive from invasive disease, predict outcome, and highlight inflammatory pathways in digestive cancers

The stromal compartment is increasingly recognized to play a role in cancer. However, its role in the transition from preinvasive to invasive disease is unknown. Most gastrointestinal tumors have clearly defined premalignant stages, and Barrett’s esophagus (BE) is an ideal research model. Supervised clustering of gene expression profiles from microdissected stroma identified a gene signature that could distinguish between BE metaplasia, dysplasia, and esophageal adenocarcinoma (EAC). EAC patients overexpressing any of the five genes (TMEPAI, JMY, TSP1, FAPα, and BCL6) identified from this stromal signature had a significantly poorer outcome. Gene ontology analysis identified a strong inflammatory component in BE disease progression, and key pathways included cytokine–cytokine receptor interactions and TGF-β. Increased protein levels of inflammatory-related genes significantly up-regulated in EAC compared with preinvasive stages were confirmed in the stroma of independent samples, and in vitro assays confirmed functional relevance of these genes. Gene set enrichment analysis of external datasets demonstrated that the stromal signature was also relevant in the preinvasive to invasive transition of the stomach, colon, and pancreas. These data implicate inflammatory pathways in the genesis of gastrointestinal tract cancers, which can affect prognosis.

DNA Methylation as an Adjunct to Histopathology to Detect Prevalent, Inconspicuous Dysplasia and Early-Stage Neoplasia in Barrett's Esophagus

Purpose: Endoscopic surveillance of Barretts esophagus is problematic because dysplasia/early-stage neoplasia is frequently invisible and likely to be missed because of sampling bias. Molecular abnormalities may be more diffuse than dysplasia. The aim was therefore to test whether DNA methylation, especially on imprinted and X-chromosome genes, is able to detect dysplasia/early-stage neoplasia. Experimental design: 27K methylation arrays were used to find genes best able to differentiate between 22 Barretts esophagus and 24 esophageal adenocarcinoma (EAC) samples. These were validated using pyrosequencing on a retrospective cohort (60 Barretts esophagus, 36 dysplastic, and 90 EAC) and then in a prospective multicenter study (98 Barretts esophagus patients, including 28 dysplastic and 9 early EAC) designed to utilize biomarkers to stratify patients according to their prevalent dysplasia/EAC status. Results: Genes (23%) on the array, including 7% of X-linked and 69% of imprinted genes, have shown statistically significant changes in methylation in EAC versus Barretts esophagus (Wilcoxon P < 0.05). 6/7 selected candidate genes were successfully internally (Pearsons P < 0.01) and externally validated (ANOVA P < 0.001). Four genes (SLC22A18, PIGR, GJA12, and RIN2) showed the greatest area under curve (0.988) to distinguish between Barretts esophagus and dysplasia/EAC in the retrospective cohort. This methylation panel was able to stratify patients from the prospective cohort into three risk groups based on the number of genes methylated (low risk: <2 genes, intermediate: 2, and high: >2). Conclusion: Widespread DNA methylation changes were observed in Barretts carcinogenesis including ≈70% of known imprinted genes. A four-gene methylation panel stratified patients with Barretts esophagus into three risk groups with potential clinical utility. Clin Cancer Res; 19(4); 878–88. ©2012 AACR.

Three-Gene Immunohistochemical Panel Adds to Clinical Staging Algorithms to Predict Prognosis for Patients With Esophageal Adenocarcinoma

PURPOSE Esophageal adenocarcinoma (EAC) is a highly aggressive disease with poor long-term survival. Despite growing knowledge of its biology, no molecular biomarkers are currently used in routine clinical practice to determine prognosis or aid clinical decision making. Hence, this study set out to identify and validate a small, clinically applicable immunohistochemistry (IHC) panel for prognostication in patients with EAC. PATIENTS AND METHODS We recently identified eight molecular prognostic biomarkers using two different genomic platforms. IHC scores of these biomarkers from a UK multicenter cohort (N = 374) were used in univariate Cox regression analysis to determine the smallest biomarker panel with the greatest prognostic power with potential therapeutic relevance. This new panel was validated in two independent cohorts of patients with EAC who had undergone curative esophagectomy from the United States and Europe (N = 666). RESULTS Three of the eight previously identified prognostic molecular biomarkers (epidermal growth factor receptor [EGFR], tripartite motif-containing 44 [TRIM44], and sirtuin 2 [SIRT2]) had the strongest correlation with long-term survival in patients with EAC. Applying these three biomarkers as an IHC panel to the validation cohort segregated patients into two different prognostic groups (P < .01). Adjusting for known survival covariates, including clinical staging criteria, the IHC panel remained an independent predictor, with incremental adverse overall survival (OS) for each positive biomarker (hazard ratio, 1.20; 95% CI, 1.03 to 1.40 per biomarker; P = .02). CONCLUSION We identified and validated a clinically applicable IHC biomarker panel, consisting of EGFR, TRIM44, and SIRT2, that is independently associated with OS and provides additional prognostic information to current survival predictors such as stage.

A systematic approach to therapeutic target selection in oesophago-gastric cancer

Objective The success of personalised therapy depends on identification and inhibition of the oncogene(s) on which that tumour is dependent. We aimed to determine whether a receptor tyrosine kinase (RTK) array could be used to select the most effective therapeutic strategies in molecularly heterogeneous oesophago-gastric adenocarcinomas. Design Gene expression profiling from oesophago-gastric tumours (n=75) and preinvasive stages (n=57) identified the active signalling pathways, which was confirmed using immunohistochemistry (n=434). RTK arrays on a cell line panel (n=14) determined therapeutic targets for in vitro cytotoxic testing. Feasibility of this personalised approach was tested in tumour samples (n=46). Results MAPK was the most frequently activated pathway (32/75 samples (42.7%)) with progressive enrichment in preinvasive disease stages (p<0.05) and ERK phosphorylation in 148/434 (34.3%) independent samples. Cell lines displayed a range of RTK activation profiles. When no RTKs were activated, tyrosine kinase inhibitors (TKIs) and a Mek inhibitor were not useful (MKN1). In lines with a dominant phosphorylated RTK (OE19, MKN45 and KATOIII), selection of this TKI or Mek in nM concentrations induced cytotoxicity and inhibited Erk and Akt phosphorylation. In cells lines with complex activation profiles (HSC39 and OE33), a combination of TKIs or Mek inhibition (in nM concentrations) was necessary for cytotoxicity and inhibition of Erk and Akt phosphorylation. Human tumours demonstrated diverse activation profiles and 65% of cases had two or more active RTKs. Conclusions The MAPK pathway is commonly activated in oesophago-gastric cancer following activation of a variety of RTKs. Molecular phenotyping can inform a rational choice of targeted therapy.

Nitric oxide-mediated invasion in Barrett's high-grade dysplasia and adenocarcinoma

Nitric oxide (NO) has been shown to induce double strand DNA breaks in Barretts oesophagus (BO) and in other cancers has a role in invasion. The specific aims of this study were to investigate whether NO can induce invasion in cells representative of different stages of Barretts progression and to determine possible underlying mechanisms. Physiological concentrations of NO that mimic luminal production of NO from dietary sources enhanced invasion in cell lines from high-grade dysplasia (GihTERT) and oesophageal adenocarcinoma (FLO) but not a non-dysplastic Barretts cell line (QhTERT). Real-time reverse transcription-polymerase chain reaction revealed that NO induced expression of matrix metalloproteinase (MMP)-1, -3, -7, -9 and -10 and tissue inhibitor of metalloproteinase (TIMP)-1, -2 and -3 in these cell lines. Furthermore, ex vivo treatment of Barretts biopsy samples with NO induced increases in MMP-1 and TIMP-1 expression, suggesting that NO enhances invasion through deregulating MMP and TIMP expression in epithelial cells. In keeping with these findings, microarray analysis and immunohistochemistry performed on biopsy samples showed enhanced expression of MMP-1, -3, -7 and -10 and TIMP-1 in the progression from non-dysplastic BO to adenocarcinoma, although this could not be directly attributed to the effect of NO. Thus, NO may play a role in Barretts carcinogenesis through deregulating MMP and TIMP expression to enhance invasive potential.

Amplification of TRIM44: Pairing a Prognostic Target With Potential Therapeutic Strategy

Background Many prognostic biomarkers have been proposed recently. However, there is a lack of therapeutic strategies exploiting novel prognostic biomarkers. We aimed to propose therapeutic options in patients with overexpression of TRIM44, a recently identified prognostic gene. Methods Genomic and transcriptomic data of epithelial cancers (n = 1932), breast cancers (BCs; n = 1980) and esophago-gastric cancers (EGCs; n = 163) were used to identify genomic aberrations driving TRIM44 overexpression. The driver gene status of TRIM44 was determined using a small interfering RNA (siRNA) screen of the 11p13 amplicon. Integrative analysis was applied across multiple datasets to identify pathway activation and potential therapeutic strategies. Validation of the in silico findings were performed using in vitro assays, xenografts, and patient samples (n = 160). Results TRIM44 overexpression results from genomic amplification in 16.1% of epithelial cancers, including 8.1% of EGCs and 6.1% of BCs. This was confirmed using fluorescent in situ hybridization. The siRNA screen confirmed TRIM44 to be a driver of the amplicon. In silico analysis revealed an association between TRIM44 and mTOR signalling, supported by a decrease in mTOR signalling after siRNA knockdown of TRIM44 in cell lines and colocalization of TRIM44 and p-mTOR in patient samples. In vitro inhibition studies using an mTOR inhibitor (everolimus) decreased cell viability in two TRIM44-amplified cells lines by 88% and 70% compared with 35% in the control cell line. These findings were recapitulated in xenograft models. Conclusions Genomic amplification drives TRIM44 overexpression in EGCs and BCs. Targeting the mTOR pathway provides a potential therapeutic option for TRIM44-amplified tumors.

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.

PWE-164 Utilizing Integrative Genomic Analysis and Proteomics to Decipher the Biology and Therapeutic Potential of Trim44 in Oesophageal Adenocarcinoma and Breast Cancer

Introduction The incidence of oesophageal adenocarcinoma (OAC) has quadrupled in the last 30 years and outcomes remain poor. We have previously identified TRIM44 as an independent prognostic gene commonly amplified in OAC and breast cancer. However, the exact biology of TRIM44 and its role in epithelial cancers remain unclear Methods Gene set enrichment analysis (GSEA) was performed on gene expression microarray data of oesophageal (n = 146) and breast cancers (METABRIC, n = 1980) to identify signalling pathways activated by TRIM44 amplification and overexpression. Mass spectrometry was used to identify binding partners of TRIM44 in both endogenous and overexpression settings. Validation of the mass spectrometry results were performed using reciprocal co-immunoprecipitations experiments Results GSEA performed on OAC samples identified 14 pathway signatures that were significantly enriched with TRIM44 overexpression. To validate these results, GSEA was performed on the METABRIC dataset and this revealed that the PI3K-AKT-mTOR signalling was the only pathway out of the 14 identified signatures to be significantly overenriched in samples with TRIM44 amplification in OAC and breast cancer (p < 0.05). Mass spectrometry of immunoprecipitated TRIM44 identified 2 novel binding partners of TRIM44 -- a ring finger protein associated with activation of c-jun and a tumour metastatic gene shown to directly activate the PI3K-AKT-mTOR signalling pathway. Validation of these two binding partners was successfully performed with endogenous co-immunoprecipitation of TRIM44 in HSC-39, a cell line with high level amplifications of TRIM44; demonstrating that both binding partners associate with TRIM44 in the endogenous setting. Conclusion Integrative genomic analysis and GSEA provided an insight into the pathways activated by TRIM44. The mTOR pathway was consistently associated with TRIM44 amplification and overexpression. A proteomics approach identified two potential mechanistic explanations how TRIM44 activates the mTOR pathway. Clinically, these findings open up the possibilities of using mTOR inhibitors or peptides disrupting TRIM44 protein interactions to treat TRIM44 amplified tumours. Disclosure of Interest None Declared.

OC-008 Defining the genetic landscape of oesophageal adenocarcinoma by next-generation sequencing

Introduction Despite its dismal prognosis and steady increase in prevalence little is known about the genetic alterations that drive oesophageal adenocarcinoma. As part of the International Cancer Genome Consortium: Oesophageal Adenocarcinoma (ICGC-OAC) we performed a pilot study, sequencing the genomes of 32 OAC samples, to assess the feasibility of initiating a large scale project sequencing a total of 500 OAC genomes. Methods A total of 56 genomes were selected for sequencing including 32 OAC genomes—16 chemotherapy-Naive, 16 chemotherapy-treated—and 24 matched normal genomes. Whole genome sequencing was performed on the Illumina Hiseq 2000 platform. Initial bioinformatic analysis, run by Illumina using the Casava pipeline, detected single nucleotide variants (SNVs), small (<50 bp) Insertion and Deletions events (INDELs) and large scale structural variants (SVs). Additionally, bioinformatic analysis of SVs was performed using a custom Perl script. To determine the specificity of the bioinformatic approach a subset of SNVs and SVs were selected for verification by Sanger capillary sequencing and PCR respectively. Results A minimum of 50-fold mappable sequence data were generated for each of the 56 genomes. 161/167 (96%) of predicted SNVs were confirmed as somatic, two were miscalled germline variants while four were undetectable in either sample. For 2/75 (3%) SVs PCR amplicons could not be generated, for 18 of 75 SVs (24%) a PCR amplicon was detectable in the normal showing them to be germline polymorphisms. The True positive rate for SV detection was therefore 73%. Comparison of SNV information across all 24 samples revealed many recurrently mutated genes. These include previously reported mutations in TP53, CDKN2A and APC among others. No genes were significantly associated with chemotherapy-treated or chemotherapy-naive samples. Conclusion Analysis of the Illumina bioinformatic pipeline suggests it is highly specific (96% true positive rate) for somatic SNVs. A true positive rate of 73% for SV detection is comparable to recent literature. Further analysis to determine the sensitivity of this pipeline is ongoing including resequencing of putatively non-mutated genes in samples sent for WGS and the application of alternative bioinformatic approaches for the calling of SNVs, INDELs and SVs. Initial analysis of the SNV data from 32 tumour genomes has revealed several recurrently mutated genes known to be altered in OAC validating the ability of our approach to detect candidate “driver” genes. Competing interests None declared.