Purpose: Transcriptomic profiling of colorectal cancer (CRC) has led to the identification of four consensus molecular subtypes (CMS1 to 4) that have prognostic value in stage II and III disease. More recently, the Colorectal Cancer Intrinsic Subtypes (CRIS) classification system has helped to define the biology specific to the epithelial component of colorectal tumors; however, the clinical value of these classification systems in the prediction of response to standard-of-care adjuvant chemotherapy remains unknown. Patients and Methods: Using samples from four European sites, we assembled a novel cohort of patients with stage II and III CRC (n = 156 samples) and performed transcriptomic profiling and targeted sequencing and generated a tissue microarray to enable integrated multiomics analyses. We also accessed data from two published cohorts of patients with stage II and III CRC: GSE39582 and GSE14333 (n = 479 and n = 185 samples, respectively). Results: The epithelial-rich CMS2 subtype of CRC benefitted significantly from treatment with adjuvant chemotherapy in both stage II and III disease ( P = .02 and P < .001, respectively), whereas the CMS3 subtype significantly benefitted in stage III only ( P = .001). After CRIS substratification of CMS2, we observed that only the CRIS-C subtype significantly benefitted from treatment with adjuvant chemotherapy in stage II and III disease ( P = .0081 and P < .001, respectively), whereas the CRIS-D subtype significantly benefitted in stage III only ( P = .0034). We also observed that CRIS-C patients with low levels of CD8⁺ tumor-infiltrating lymphocytes were most at risk for relapse in both stage II and III disease (log-rank P = .0031; hazard ratio, 12.18 [95% CI, 1.51 to 98.58]). Conclusion: Patient stratification using a combination of transcriptional subtyping and CD8 immunohistochemistry analyses is capable of identifying patients with poor prognostic stage II and III disease who benefit from adjuvant standard-of-care chemotherapy. These findings are particularly relevant for patients with stage II disease, where the overall benefit of adjuvant chemotherapy is marginal.

Drug resistance limits the effectiveness of oesophageal adenocarcinoma (OAC) chemotherapies, leading to a poor prognosis for this disease. Elucidation of the underlying resistance mechanisms is key to enabling the identification of more effective treatments. This study, therefore, aims to identify novel therapeutic and/or chemotherapy sensitising drug targets in OAC. Transcriptional data from a cohort of 273 pre-treatment OAC biopsies, from patients who received neoadjuvant chemotherapy followed by surgical resection, were analysed using gene set enrichment analysis (GSEA) to determine differential gene expression between responding and non-responding OAC tumours. From this, 80 genes were selected for high-throughput siRNA screening in OAC cell lines with or without standard chemotherapy treatment. In parallel, cell viability assays were performed using a panel of FDA-approved drugs and combination index (CI) values were calculated to evaluate drug synergy with standard chemotherapy. Mechanisms of synergy were investigated using western blot, propidium iodide flow cytometry, and proliferation assays. Taken together, the screens identified that targeting Src, using either siRNA or the small molecule inhibitor dasatinib, enhanced the efficacy of chemotherapy in OAC cells. Further in vitro functional analysis confirmed Src inhibition to be synergistic with standard OAC chemotherapies, 5-fluorouracil (5-FU), and cisplatin (CDDP). In conclusion, a compound screen together with a functional genomic approach identified Src as a potential chemosensitising target in OAC, which could be assessed in a clinical study for poor prognosis OAC patients.

Introduction: Five-year survival rates for oesophageal adenocarcinoma (OAC) remain poor at just 15%. The development of drug-resistance limits the effectiveness of current chemotherapies, so the discovery of underlying resistance mechanisms and novel agents to target these pathways is crucial. Materials and Methods: 273 pre-treatment OAC biopsies were transcriptionally profiled using the Almac Diagnostics Xcel array™. The Broad Institute's Gene Set Enrichment Analysis (GSEA) software was used to identify pathways enriched in non-responders to chemotherapy compared to responders. A focused siRNA screen of 80 target genes identified from the GSEA results was then devised and completed, highlighting the role of Src in chemotherapy non-responders. An Enzo compound screen of FDA-approved drugs was carried out in OE33 and FLO-1 cell lines in combination with cisplatin, and cell viability was measured by CellTiter Glo. Src inhibition using dasatinib and saracatinib was assessed with MTT viability assays, western blotting and flow cytometry to investigate the mechanisms of action. CI values were generated to assess synergy. Results: GSEA identified pathways associated with resistance to chemotherapy in OAC. Candidate genes were selected according to predefined criteria and 80 genes were taken forward in a siRNA screen to study the effects on cell viability of gene silencing alone or in combination with cisplatin or 5-FU. Twelve genes were found to have an additive effect with chemotherapy to improve chemo-efficacy across an OAC cell line panel, including Src. A high-throughput drug screen highlighted a number of novel compounds that displayed additive effects when combined with cisplatin in OAC cell lines including dasatinib, a Src inhibitor. Src was then validated in an in-vitro OAC setting, investigating the effects of Src inhibition using siRNA, dasatinib and saracatinib. In-vitro combination experiments showed that combining Src inhibitors with traditional chemotherapies provides synergistic effects. Conclusions: Using a functional genomic approach Src was identified as a novel target associated with reduced cell viability following siRNA-mediated knockdown. By the use of small molecule inhibitors, the results of the siRNA screen were reinforced, verifying the clinical potential and synergistic effects of targeting Src alongside traditional chemotherapy treatments. Citation Format: Niamh Helen McCabe, Leanne Stevenson, Enya Scanlon, Oliver Keminer, Richard D. Kennedy, Bjoern Windshuegel, Jaine K. Blayney, Richard C. Turkington. Identification of Src's role in governing chemotherapy resistance in oesophageal adenocarcinoma through functional genomic and high-throughput drug screening approaches [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2999.

E15142 Background: Ulcerative colitis (UC), is a known risk-factor for colorectal cancer (CRC). 10% of UC-patients develop UC-specific CRC yet there are currently no biomarkers for early detection or understanding of drivers of disease. Unresolved inflammation is key in UC-CRC development, yet the aetiology is unclear. It is unknown if UC-CRC falls into a specific consensus molecular subgroup in the CMS classification (Guinney et al 2015). This bioinformatics approach aimed to identify patients at-risk of developing UC-CRC, whilst informing biological and clinical understanding of the disease. Methods: We used publicly available UC data and in-silico, cross platform, class label transfer (GECA) to determine mRNA similarity between UC/UC-CRC and sporadic CRC (sCRC) and to identify a UC-predominant CMS. Pathogenic mutations were identified for each CMS as well as gene set enrichment and survival analysis to determine UC-CRC behaviour comparative to sCRC. Transcriptional relationships were assessed via unsupervised and semi-supervised clustering by immune-signatures and scores to determine molecular patterns between phenotypes. We derived a gene list from UC data informed by clustering to stratify patients based on risk to UC-CRC progression. Results: UC-CRC featured predominantly in the NA group, with 3 attributed pathogenic mutations, an oxidative stress environment and neuroendocrine dysregulation. The subgroup had UC-like clinical features; younger age, distal tumour location and TP53 mutations along with the highest rate of cancer recurrence (~20%) and a unique cellular composition in CMS being highly immune and stromal. Importantly this immune signal was distinct from the interferon type signalling observed in CMS1 and was not associated with PD-L1 activation. Clustering revealed a loss of IFN-type gene expression in colon tissue as UC progressed to UC-CRC. Conclusions: The NA group was thought to be unclassifiable samples between CMS however, analysis shows UC-CRC enrichment with defined biological characteristics. We have identified novel biology that may inform the risk of developing UC-CRC and guide potential preventative and therapeutic approaches.

Background: The Dual Erb B Inhibition in Oesophago-gastric Cancer (DEBIOC) trial reported an acceptable safety profile for neoadjuvant oxaliplatin and capecitabine (Xelox) +/- AZD8931 in oesophageal adenocarcinoma (OAC) but limited efficacy. We evaluated the impact of neoadjuvant Xelox +/- AZD8931, a novel small-molecule inhibitor with equipotent activity against epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER)2 and HER3, on biological pathways using a unique software-driven solution. Patients and methods: Transcriptomic profiles from 25 pre-treatment formalin-fixed paraffin-embedded OAC biopsies and 18 matched resection specimens, treated with Xelox + AZD8931 (n = 16) and Xelox alone (n = 9), were analysed using the Almac claraT total mRNA report analysing 92 gene signatures, 100 unique single-gene drug targets and 7337 single genes across 10 hallmarks of cancer. Gene-set enrichment analysis (GSEA) was utilised to investigate pathways governing pathological response. Tumour-infiltrating lymphocytes (TILs) were assessed digitally using the QuPath software. Results: Hierarchical clustering identified three molecular subgroups classified by activation of innate immune signalling. The immune-high subgroup was associated with HER2 positivity, increased pathological response and a marked reduction in immune signalling and TILs following neoadjuvant therapy. The immune-low cluster was predominantly HER2/EGFR-negative, and EGFR positivity was associated with the immune-mixed subgroup. Treatment with neoadjuvant therapy induced common resistance mechanisms, such as angiogenesis and epithelial emesenchymal transition signalling, and a reduction in DNA repair signatures. Addition of AZD8931 was associated with reduction of expression of EGFR, HER2 and AKT pathways and also promoted an immunosuppressive microenvironment. GSEA showed that patients with a pathological response to treatment had increased immune signalling, whereas non-responders to neoadjuvant therapy were enriched for nucleotide repair and cellular growth through the action of E2F transcription factors. Conclusion: OAC may be subdivided into three immune-related subgroups which undergo modulation in response to neoadjuvant therapy with marked suppression of the immune microenvironment in HER2-positive/immune-high tumours.