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The prognostic benefit of primary tumour and lymph node (LN) downstaging after neoadjuvant chemotherapy for oesophageal adenocarcinoma (OAC) is well described. However, there is no robust evidence regarding the prognostic effect of pathological LN regression despite emerging evidence of a discrepancy, in some patients, between tumour regression grade in the primary tumour and response in regional LNs. The aim of this study was to investigate the relationship between pathological LN regression, tumour recurrence and survival. Retrospective, multicentre cohort study including 763 patients with OAC treated with neoadjuvant chemotherapy followed by surgery at 5 high-volume tertiary referral centres in the United Kingdom. Tumour regression was assessed in the primary tumour (Mandard) and LNs retrieved from oesophagectomy specimens. Patients were classified as LN negative (no evidence of tumour or regression in any LN), complete LN-responders (evidence of regression ≥1 LN, no residual tumour in any LN), partial LN-responders (evidence of regression ≥1 LN with residual tumour ≥1 LN) and LN non-responders (no or minimal regression in any LN). Survival analysis was performed using Kaplan-Meier and Cox regression. Overall, 243 (31.8%) patients were classified as LN negative, 62 (8.1%) as complete LN-responders, 155 (20.3%) as partial LN-responders and 303 (39.7%) as LN non-responders. Some patients had a LN response in the absence of a response in the primary tumour (97/431, 22.5%). Multivariable Cox regression survival analysis (adjusting for age, gender, chemotherapy regimen, clinical stage, tumour grade, lympho-vascular invasion and primary tumour response) demonstrated improved overall survival in complete LN-responders (Hazard ratio (HR) 0.37 95% confidence interval (CI) 0.24-0.58), partial LN-responders (HR 0.70 95% CI 0.55-0.89) and LN negative patients (HR 0.34 95% CI 0.26-0.44) compared to LN non-responders. In this cohort of patients with OAC treated with neoadjuvant chemotherapy prior to surgical resection, LN regression was a strong prognostic factor independent of primary tumour response, which was discordant in a significant number of patients. Complete LN-responders had equivalent survival to those with negative LN. Complete and partial LN-responders had better survival than LN non-responders. Evaluation and documentation of LN regression should be considered during the standard pathological reporting of oesophagectomy specimens.

e15535 Background: The incidence of Esophageal Adenocarcinoma (EAC) has risen in the western world but response rates to chemotherapy are low and survival is poor. Increased molecular understanding is needed to develop novel treatments. Methods: Transcriptional profiling of 274 treatment naïve EAC biopsies was performed using the Almac Diagnostics Xcel array™. All patients received platinum-based neo-adjuvant chemotherapy prior to surgical resection at four United Kingdom centers between 2004-2012. Semi-supervised clustering was performed followed by functional enrichment using DAVID. Cluster membership was assessed for independence of prognostic factors using Cox proportional hazards. Candidate targets were identified by siRNA screening in OE33 cells. Treatment with the survivin inhibitor, YM155 in EAC cell lines was also assessed. Results: Semi-supervised hierarchical clustering identified two groups with significant differences in RFS [HR = 0.54 (0.29-0.99), p = 0.05] and OS [HR = 0.52 (0.28-0.96), p = 0.04]. There were significant associations between the clusters and both nodal and TNM downstaging but not with pathological response. The PI3K-AKT, p53, tight junction and HIF-1 signaling pathways were upregulated in the poor prognostic group. Eighty-four genes were selected and taken forward in a functional genomic siRNA screen. Twenty-seven genes showed a significant reduction in viability following siRNA-mediated knockdown and further verification resulted in twelve candidate genes. Finally, target knockdown in seven EAC cell lines resulted in four interrelated hits- BIRC5, JAK1, OSMR and SLC2A1. Knock down of BIRC5 (Survivin) induced apoptosis, as evidenced by PARP cleavage, in both the parental OE33 and cisplatin-resistant OE33CDDPR cell lines. YM155, a survivin inhibitor, is shown to induce apoptosis at nanomolar concentrations across a panel of parental and cisplatin-resistant EAC cell lines and further mechanistic work is ongoing. Conclusions: We have performed clustering of a large transcriptomic dataset and defined a poor prognostic group of EAC patients. We identified Survivin (BIRC5) as a mediator of cisplatin resistance in EAC and a potential novel drug target. Further pre-clinical and clinical work to assess the benefit of survivin inhibition in EAC is warranted.

4026 Background: Current strategies to guide the selection of neo-adjuvant or adjuvant therapy in esophageal and gastric adenocarcinomas (EAC/GAC) are inadequate. We assessed a clinically validated 44 gene DNA Damage Response Deficiency (DDRD) assay to predict prognosis following neo-adjuvant DNA damaging chemotherapy (CT) in EAC and adjuvant CT or chemo-radiotherapy (CRT) in GAC. Methods: Transcriptional profiling of 273 formalin fixed paraffin embedded pre-treatment endoscopic EAC biopsies was performed using the Almac Diagnostics Xcel array. All EAC patients were treated with cisplatin-based neo-adjuvant chemotherapy followed by surgical resection between 2003 and 2014 at four UK centers in the OCCAMS consortium. Further validation was performed using a publically available dataset of 270 resected gastric cancers treated with adjuvant platinum-based CT, CRT or surgery alone at the Samsung Medical Centre, Seoul, Korea. The association between the DDRD score and prognosis was assessed by Kaplan-Meier analysis and Cox Proportional Hazards regression. Results: A total of 66 EAC samples (24%) were characterized as DDRD positive with the remaining 207 samples (76%) being DDRD negative. DDRD assay positivity was associated with improved DFS (HR 0.58; 95% CI 0.36-0.93; p = 0.024) and OS (HR 0.56; 95% CI 0.34-0.92; p = 0.023) following multivariate analysis. DDRD positive patients had a higher pathological response rate (p = 0.033) and a higher rate of loco-regional versus distant relapse (30% vs 20%; p = 0.013). For GAC, 132 samples (49%) were characterized as DDRD positive with the remaining 138 (51%) being DDRD negative. DDRD positivity was associated with improved DFS (HR 0.48; 95% CI 0.25-0.96; p = 0.037) following D2 gastrectomy and adjuvant CT or CRT. DDRD status was not associated with DFS in the surgery alone cohort (HR 0.87; 95% CI 0.55-1.38; p = 0.562). Conclusions: The DDRD assay is strongly predictive of benefit from DNA damaging neo-adjuvant CT and esophagectomy in EAC and gastrectomy and CT/CRT in GAC and can be applied to routine diagnostic material.

4023 Background: Programmed Death-1 Receptor (PD-1) and its ligand (PD-L1) downregulate T cell activation and suppress tumor killing. This study investigated the role of PD-L1 and tumor infiltrating lymphocytes (TILs) in response to neo-adjuvant therapy and prognosis in esophageal adenocarcinoma (EAC). Methods: Transcriptional profiling of 273 formalin fixed paraffin embedded pre-treatment endoscopic EAC biopsies was carried out using the Almac Diagnostics Xcel array and the expression levels of PD-L1 probesets corresponding to protein encoding extracted. Response was assessed by tumor regression grade (TRG; score ≤ 2 = response). Immunohistochemistry (IHC) for PD-L1 and CD8 was performed in matched resection specimens from 135 patients. All EAC patients were treated with cisplatin-based neo-adjuvant chemotherapy followed by surgical resection between 2003 and 2014 at four UK centers as part of the OCCAMS consortium. Associations between expression, protein levels and TRG were assessed by Kruskal-Wallis, Mann-Whitney Unpaired, Spearman rank correlation or chi-squared tests. Survival analysis was performed using Cox Proportional Hazards regression. Results: High PD-L1 gene expression in the pre-chemotherapy biopsies was associated with pathological response (TRG ≤ 2; p = 0.02) following neo-adjuvant chemotherapy. PD-L1 ( > 5%) was expressed in the tumor or stromal cells in 4% and 15% of resection specimens respectively. PD-L1 gene and IHC expression ( > 5%) were closely associated between the biopsies and both the tumor (p = 0.032) and stroma (p = 0.019) of the matched resection specimens. Patients with PD-L1 IHC positivity in tumor cells demonstrated improved relapse-free survival (HR 0.314; 95% CI 0.099-0.997; p = 0.049) and positive stromal PD-L1 IHC staining correlated with pathological response (p = 0.05). Biopsy gene expression of PD-L1 and CD8 was closely associated (p = 0.024) and the presence of CD8+ TILs in the microenvironment strongly correlated with tumor (p < 0.001) and stromal (p < 0.001) PD-L1 positivity. Conclusions: High PD-L1 expression in the pre-treatment biopsies in EAC is predictive of response to neo-adjuvant chemotherapy and may aid selection of conventional and immune-targeted agents.

ObjectiveCurrent strategies to guide selection of neoadjuvant therapy in oesophageal adenocarcinoma (OAC) are inadequate. We assessed the ability of a DNA damage immune response (DDIR) assay to predict response following neoadjuvant chemotherapy in OAC.DesignTranscriptional profiling of 273 formalin-fixed paraffin-embedded prechemotherapy endoscopic OAC biopsies was performed. All patients were treated with platinum-based neoadjuvant chemotherapy and resection between 2003 and 2014 at four centres in the Oesophageal Cancer Clinical and Molecular Stratification consortium. CD8 and programmed death ligand 1 (PD-L1) immunohistochemical staining was assessed in matched resection specimens from 126 cases. Kaplan-Meier and Cox proportional hazards regression analysis were applied according to DDIR status for recurrence-free survival (RFS) and overall survival (OS).ResultsA total of 66 OAC samples (24%) were DDIR positive with the remaining 207 samples (76%) being DDIR negative. DDIR assay positivity was associated with improved RFS (HR: 0.61; 95% CI 0.38 to 0.98; p=0.042) and OS (HR: 0.52; 95% CI 0.31 to 0.88; p=0.015) following multivariate analysis. DDIR-positive patients had a higher pathological response rate (p=0.033), lower nodal burden (p=0.026) and reduced circumferential margin involvement (p=0.007). No difference in OS was observed according to DDIR status in an independent surgery-alone dataset.DDIR-positive OAC tumours were also associated with the presence of CD8+ lymphocytes (intratumoural: pConclusionThe DDIR assay is strongly predictive of benefit from DNA-damaging neoadjuvant chemotherapy followed by surgical resection and is associated with a proinflammatory microenvironment in OAC.