Alterations in the Ca2+ toolkit in oesophageal adenocarcinoma

Aim: To investigate alterations in transcription of genes, encoding Ca2+ toolkit proteins, in oesophageal adenocarcinoma (OAC) and to assess associations between gene expression, tumor grade, nodal-metastatic stage, and patient survival. Methods: The expression of 275 transcripts, encoding components of the Ca2+ toolkit, was analyzed in two OAC datasets: the Cancer Genome Atlas [via the University of Alabama Cancer (UALCAN) portal] and the oesophageal-cancer, clinical, and molecular stratification [Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS)] dataset. Effects of differential expression of these genes on patient survival were determined using Kaplan-Meier log-rank tests. OAC grade- and metastatic-stage status was investigated for a subset of genes. Adjustment for the multiplicity of testing was made throughout. Results: Of the 275 Ca2+-toolkit genes analyzed, 75 displayed consistent changes in expression between OAC and normal tissue in both datasets. The channel-encoding genes, N-methyl-D-aspartate receptor 2D (GRIN2D), transient receptor potential (TRP) ion channel classical or canonical 4 (TRPC4), and TRP ion channel melastatin 2 (TRPM2) demonstrated the greatest increase in expression in OAC in both datasets. Nine genes were consistently upregulated in both datasets and were also associated with improved survival outcomes. The 6 top-ranking genes for the weighted significance of altered expression and survival outcomes were selected for further analysis: voltage-gated Ca2+ channel subunit α 1D (CACNA1D), voltage-gated Ca2+ channel auxiliary subunit α2 δ4 (CACNA2D4), junctophilin 1 (JPH1), acid-sensing ion channel 4 (ACCN4), TRPM5, and secretory pathway Ca2+ ATPase 2 (ATP2C2). CACNA1D, JPH1, and ATP2C2 were also upregulated in advanced OAC tumor grades and nodal-metastatic stages in both datasets. Conclusions: This study has unveiled alterations of the Ca2+ toolkit in OAC, compared to normal tissue. Such Ca2+ signalling findings are consistent with those from studies on other cancers. Genes that were consistently upregulated in both datasets might represent useful markers for patient diagnosis. Genes that were consistently upregulated, and which were associated with improved survival, might be useful markers for patient outcome. These survival-associated genes may also represent targets for the development of novel chemotherapeutic agents.

P-OGC39 The smell of oesophageal adenocarcinoma: opportunities for tests and treatments

Background Exhaled breath analysis is a promising approach for oesophageal adenocarcinoma (OAC) early detection. The biomarkers of interest are low molecular weight metabolites including volatile aldehydes. In this translational study we investigated whether these metabolites originated from a tumoral source, and how this might impact the diagnosis and treatment of OAC patients. Methods The investigative strategy was directed by an unbiased informatics screen of metabolic reprogramming in OAC, and validated using complimentary gene expression assays (n = 638, including controls). Mass spectrometric methods were used to quantify corresponding metabolites and putative source compounds at a tissue level (n = 158), and also in exhaled breath for correlative purposes. Targeted in vitro experiments were performed to demonstrate the cause and effect of the proposed model of metabolic reprogramming in OAC. Results The unbiased screen and subsequent validation found that reduced aldehyde detoxification is an OAC hallmark. In vitro and in vivo this was associated with endogenous aldehyde accumulation. OAC tissue was generally enriched for volatile aldehydes, including the genotoxins formaldehyde, acetaldehyde, 4-hydroxy-2-nonenal and 2-butenal, and the exhaled biomarker decanal (all P < 0.0001). Decanal concentrations correlated with exhaled concentrations. Considering potential aldehyde sources, the OAC phospholipidome was characterised by desaturated and longer lipid acyls, and these spontaneously generated biomarker aldehyde species at ambient conditions. Enriched genotoxic aldehydes were detectable in base-pairing positions in DNA; this genotoxicity was therapeutically targetable with aldehyde scavengers in vitro. Conclusions These data support a model for enriched exhaled aldehydes based on increased production from an altered lipid phenotype, and reduced detoxification. Some aldehydes are non-reactive and thus support non-invasive detection. Others react with DNA and increase local genotoxicity; this process is druggable. These findings have implications for OAC early diagnosis and chemoprevention.

O-OGC06 Genomic analysis of response to neoadjuvant chemotherapy in oesophageal adenocarcinoma

Background Oesophageal adenocarcinoma (OAC) is the ninth most common cancer worldwide, with an estimated mortality of over 500,000 deaths yearly. Neoadjuvant chemotherapy (NAC) followed by surgery is the standard of care (SOC) for locally advanced OAC. Although almost all patients receive chemotherapy as SOC, fewer than 20% obtain a clinically meaningful response and benefit before surgery. The OAC genome is complex and heterogeneous between patients, and it is not yet understood whether specific mutational patterns may result in chemotherapy sensitivity or resistance. Methods To identify associations between genomic events and response to NAC in OAC, a comparative genomic analysis was performed in 65 patients using whole-genome sequencing. We defined response to NAC using Mandard Tumour Regression Grade (TRG), with responders classified as TRG1-2 (n = 27) and non-responders classified as TRG4-5 (n = 38). Results We report a higher non-synonymous mutation burden in responders (median 2.08/Mb vs 1.70/Mb, P = 0.036) and elevated copy number variation (CNV) in non-responders (282 vs 136/patient, P<0.001). We identified CNVs unique to each group, with cell cycle (CDKN2A, CCND1), c-Myc (MYC), RTK/PIK3 (KRAS, EGFR) and gastrointestinal differentiation (GATA6) pathway genes being specifically altered in non-responders. Of particular interest was the identification of the Neuron Navigator-3 (NAV3), a known tumour suppressor downstream of EGFR, which was mutated exclusively in non-responders with a frequency of 22%. Conclusions Our work characterises genetic features and mutations that are uniquely associated with response to NAC. We envision a treatment pipeline that incorporates driver mutation profiling in OAC, combining response prediction with targeted therapies to enhance response to NAC and improve survival outcomes.

P-OGC44 Multi-omic cohort study of Barrett’s oesophagus reveals structural variation and retrotransposon activity to occur early in cancer evolution

Background Barrett’s oesophagus (BE) is the main risk factor for the development of oesophageal adenocarcinoma (OAC), yet few patients ever go on to progress to cancer. The acquisition of events during the metaplasia-dysplasia-cancer sequence is poorly characterised. We present a large, unbiased, multi-omics analysis of a cross-sectional cohort of pre-cancer samples, with the aim of providing a comprehensive insight into the diversity and molecular changes driving the disease to cancer. Methods We generated and integrated the genomic (50x), transcriptomic and epigenomic (850K EPIC array) landscapes of snap-frozen endoscopic biopsies from 146 patients with a range of outcomes (27 long-standing non-dysplastic; 12 prior to progression to dysplasia; 14 low-grade; 25 high-grade; 21 intramucosal carcinoma; 47 cases of BE taken adjacent to OAC) and 642 person years of follow-up. All biopsies were reviewed independently by 3 pathologists and had associated annotation with detailed clinical information. Results The total number of structural variants (SV) captured the most variance between samples. Complex SVs and LINE-1 retrotransposon activity were observed even before dysplasia had developed and increased with progression. Increasing SV burden was associated with chromothripsis (12%, 18/146) and breakage-fusion bridges (BFBs; 8%, 13/146). In more than 50% of these, the BFBs were in chromosome 17, harbouring the oncogenes ERBB2 and CDK12, for which expression was significantly higher. With progression there was increased expression of genes related to cell-cycle checkpoint, DNA repair and chromosomal instability, and the epigenetic silencing of genes in WNT-signalling and cell-cycle pathways. Conclusions Genomic complexity occurs very early in the natural history of BE and increasing genomic instability appears to tip the balance towards cancer. This may inform the potential for progression to cancer beyond the clinically discernible phenotype. Efforts to better understand the triggers for chromosomal breakages and rearrangements that underly progression will aid clinical prediction and prevention strategies.

P-OGC08 Translational Insights from the Dual ErbB Inhibition in Oesophago-gastric Cancer (DEBIOC) Clinical Trial - A Bioinformatic Analysis

Background Oesophageal Adenocarcinoma (OAC) incidence in the Western-world has increased markedly over 30 years. 5-year survival rates for patients remains below 20% with dismal response to neo-adjuvant or perioperative chemotherapy for operable tumours. The Dual ErbB Inhibition in Oesophago-gastric Cancer (DEBIOC) clinical trial assessed efficacy of combined oxaliplatin and capecitabine (Xelox) with dual ErbB inhibitor AZD8931 in providing additional benefit to operable patients compared to Xelox alone. We utilised a bioinformatic approach combing Almac Clara-T Transcriptional Discovery software with unsupervised machine learning methods to unveil translational clinical potential and biological insights from DEBIOC patient biopsy and resection specimens. Methods Using microarrays of DEBIOC patient specimens with documented clinical observations, we combined unsupervised machine learning techniques with state-of-the-art Almac Clara-T software to assess transcriptional changes between treatment types regarding the 10 hallmarks of cancer, characterised by representative gene-expression signatures and scores. These methods were employed to identify possible mechanisms of treatment resistance, evaluate changes in the tumour-microenvironment and determine clinically significant molecular subgroups in OAC. Differential expression and pathway analytics were used to describe signalling dissimilarities between clusters from unsupervised analysis and phenotypes respective to hallmarks of cancer, with alignment of sensitivities to single-gene drug targets for subgroups of interest. Results Unsupervised clustering analysis of biopsy specimens, resulted in the identification of two robust subgroups pre-treatment in OAC, determined to be significantly associated with the prediction of Mandard Score (Tumour Regression Grade 1-5) post-treatment (fishers exact p < 0.05). Differential expression analysis revealed distinguishing biology between subtypes and noted increased ErbB signalling in non-responding patients in addition to increased PI3K signalling, highlighting a potential mechanism of resistance to dual ErbB inhibition (nominal p-value <0.05, FDR p-value <0.2). Semi-supervised clustering revealed hallmark-specific-phenotypes associated with clinical observations including lymph node involvement, EGFR FISH classification, vascular invasion and progression events at BH adjusted p-values <0.05. Conclusions Our analysis has revealed translational insights into possible mechanisms of drug resistance as well as cancer hallmark-specific phenotypes significantly associated with clinico-pathological factors during the DEBIOC clinical trial. Continued analysis into resulting phenotypes and clusters combined with the alignment of single gene drug target sensitivities is anticipated to reveal novel molecular pathways driving phenotypic differences in an effort to further inform biological understanding and improve treatment response and survival outcomes in OAC patients.

P-OGC32 Copper-dependent cell death is a cancer specific vulnerability in oesophageal adenocarcinoma and provides the opportunity for future biomarker-stratified clinical trials

Background Oesophageal adenocarcinoma (OAC) is of increasing global concern due to increasing incidence, lack of effective treatments, and poor prognosis. Therapeutic target discovery and clinical trials have been hindered by the heterogeneity of the disease, dominance of large-scale genomic rearrangements, and lack of driver mutations. We have profiled small-molecule compounds using an innovative high content imaging assay in a panel of transformed and non-transformed oesophageal cell lines to identify OAC-specific cytotoxic compounds, new therapeutic targets, potential drug repurposing opportunities, and chemical starting points for the treatment of OAC. Methods We have comprehensively profiled 19,555 small-molecule compounds using an innovative high content assay to quantify 1000’s of subcellular imaging features to capture important phenotypes missed by standard approaches. Prioritised molecules then underwent functional, transcriptomic, and metabolomic characterisation across panels of oesophageal cell lines and patient-derived organoids for the identification of OAC-specific drug mechanisms. Results We identified 72 lead compounds as exhibiting OAC-specific cytotoxicity and characterised three of the most potent and selective compounds in depth, each of different proposed classes and chemical structures. Using several orthogonal methods we uncovered a unified mechanism of action and a targetable vulnerability in OAC involving copper-dependent cell death. Strikingly no phenotypic effects or changes in gene-expression were observed following treatment with these compounds in non-transformed oesophageal cell lines or normal gastric organoids providing support for this mechanism as a cancer-specific phenomenon. Conclusions We have applied high content imaging, transcriptomic and metabolomic analyses to reveal a unique vulnerability in OAC. We have defined a unified mechanism of OAC-specific copper-dependent cell death for the three highly potent compounds. Finally, through the integration of transcriptomic and metabolomics analyses we gained insight into drug sensitivity and provide the basis for a future biomarker-stratified clinical trial of these drugs in OAC.

P-OGC06 The effect of surgical complications on long-term prognosis following oesophagectomy for oesophageal cancer

Background Globally, oesophageal cancer incidence continues to increase. In recent years, surgical and oncological advancements have increased survival rates. Despite this, survival remains <50% at five-years for patients treated with curative oesophagectomy. Previous data has suggested post-operative complications may play a role in long-term increased mortality in oesophageal cancer patients. This study aimed to examine the effect of adverse in-hospital events following oesophagectomy on the long-term prognosis for oesophageal cancer, including assessing the effect of cumulative complication burden using data from a single high-volume academic unit in the UK. Methods Retrospective analysis of patients undergoing oesophagectomy for oesophageal adenocarcinoma or squamous cell carcinoma was performed to assess the relationship between in-hospital events and long-term survival. Analysis was limited to patients who survived to 90 days post-oesophagectomy (n = 380). Complications were graded according to the Clavien-Dindo (CD) classification and the Comprehensive Complication Index (CCI). Survival was estimated using Kaplan Meier survival curves and multivariate cox-regression, adjusting for variables known to influence survival. The absolute magnitude of effect of complications on survival was assessed using the risk-adjusted population attributable fraction (PAF), which estimates the percentage improvement in survival if specified complications were removed. Results Complications occurred in 251 patients (66.1%). ≥CD3a complications (HR1.65, 95%CI 1.15-2.38, p < 0.010) and unplanned critical care admissions (HR2.24, 95%CI 1.45-3.46, p < 0.001) were independently associated with worse prognosis whereas pulmonary complications and anastomotic leak were not. A CCI >30 was the optimum cut-point for OS (HR1.94, 95%CI 1.36-2.78, p < 0.001), and after weighting to remove confounding bias median survival was shorter with CCI>30 (28vs72 months, p < 0.001). There was no difference in median survival when CCI>30 occurred from major or multiple minor complications (31 vs 21 months, p = 0.096). The risk adjusted PAF for CCI>30 was 8.5% (95%CI 3.6-13.1%). Conclusions Long-term survival following oesophagectomy for oesophageal cancer is significantly affected by major complications and unplanned critical care admissions. The cumulative effect of multiple post-operative minor complications is comparable to the effect of major complications on long-term survival from oesophageal cancer, and cause a substantial number of potentially preventable deaths, even in patients who survive to discharge.

P-OGC68 Prophylactic Endoluminal Vacuum Therapy (EVT) following oesophagectomy

Background Endoluminal vacuum therapy (EVT) is an emerging treatment strategy for the management of anastomotic leaks following oesophagectomy. However, patients are often critically unwell with mediastinitis and established sepsis by the time the leak is diagnosed. This results in a protracted recovery period regardless of the effectiveness of EVT in treating the leak. Prophylactic EVT to protect the anastomosis following oesophagectomy may reduce the incidence of anastomotic leak, and/or mediastinitis and sepsis if the anastomosis does fail. We report the outcomes of two patients considered high risk for anastomotic leak who were managed with prophylactic EVT following esophagectomy for cancer. Methods Two patients received prophylactic EVT following oesophagectomy between May and July 2021. The patients were considered high risk for anastomotic leak due to technical concerns with, or complications during, the operation. In both cases the oesophagogastric anastomosis (OGA) was fashioned with a circular stapler. The endoluminal vacuum device (EVD) was constructed using an 18F nasogastric tube and a piece of open cell foam, and placed intraluminally across the anastomosis under endoscopic guidance at the time of surgery. Continuous negative pressure (125mmHg) was applied. Information relating to treatment and outcome was recorded prospectively. Results Patient-1, a 72-year-old female, ASA 2, underwent minimally invasive oesophgectomy for an adenocarcinoma at the gastro-oesophageal junction. After creating the stapled OGA, inspection revealed the proximal (oesophageal) tissue doughnut was complete but attenuated. Patient 2, a 67-year-old male, ASA 3, underwent a hybrid Ivor Lewis oesophgectomy for a lower 1/3 oesophageal adenocarcinoma. Surgery was complicated by significant intra-abdominal bleeding requiring blood transfusion and pressor support. In both cases, endoscopic assessment of the anastomosis following removal of the prophylactic EVD was performed day seven post-operatively. The anastomoses were healthy with no evidence of a leak, dehiscence, or early stricture formation. Conclusions In this limited case series, prophylactic EVT of the OGA following oesophagectomy was delivered safely with no complications related to insertion of the EVD or delivery of EVT. This intervention should be considered in cases where the risk of anastomotic leak is high. An intraluminal EVD situated across the OGA may minimise the extent of extraluminal contamination, and the systemic consequences of sepsis associated with this, should an anastomotic breakdown occur. Further studies are required to determine the safety of prophylactic EVT following oesophagectomy, and whether this improves surgical outcomes by reducing the incidence and impact of anastomotic leaks.

P-OGC34 Pandemic paradigms: Early outcomes of radical chemoradiotherapy for patients with operable oesophageal and oesophago-gastric junctional adenocarcinoma

Background The peak waves of the COVID pandemic necessitated a paradigm shift in surgical management of patients with oesophageal adenocarcinoma due to both pressure on services and high mortality rates for those with COVID undergoing surgery. The Association of Upper GI Surgeons (AUGIS) guidance on treating Upper gastrointestinal cancers in the COVID era made suggestions to treat operable adenocarcinomas using definitive/consolidation chemoradiation (DCRT) over standard neo-adjuvant chemotherapy (NAC) and our unit altered practice accordingly for a cohort of patients. For affected patients we monitored and audited clinical outcomes and the initial results from this are presented here. Methods Patients with oesophageal or oesophago-gastric junctional (O/OGJ) adenocarcinoma with potentially curative disease where initial management was altered from a treatment path which would have included surgery (with or without neoadjuvant therapy) to DCRT discussed at our regional multidisciplinary team (MDT) meeting between 1st February-1st June 2020 were included. Patient demographics, investigations, treatment given and clinical outcomes were prospectively recorded. Results 31 Patients with operable adenocarcinoma of O/OGJ had treatment altered to DCRT (mean age 65.4, [range 43 – 79]), 28 (90%) Male. 1 patient deteriorated prior to starting, leaving 30 who completed DCRT. Of these 4 patients had already had NAC prior to DCRT. Follow up was for a median of 8 (range 4-8) months following start of treatment. Post- vs pre-treatment FDG-PET imaging demonstrated a significant reduction in the mean maximum standardized uptake value (SUVmax) (p = 0.003, Sign test), in all but 3 patients. 11 patients had DCRT alone, (all alive at the time of data collection), of whom 3 patients had no sign of tumour. 19 (56%) patients proceeded to salvage oesophagectomy at a median of 15(range 10-25) weeks after completion of DCRT. 42% of these patients had a complete pathological response to treatment. There was a 5% perioperative mortality rate for this group and 1 patient was found to be unresectable on the day of surgery. At the time the data was reviewed overall survival of the entire cohort was 91%, 56% of whom had no sign of residual or recurrent disease. Conclusions A disease free survival of 56% compares poorly with the literature at the 3-month interval. The long-term follow-up of these patients will only be apparent in the coming months and years. This data does not support the use of this modality in the future and alternate treatment plans should be devised for future pandemics.