Background & AimsEffective therapeutic approaches are urgently required to tackle the alarmingly poor survival outcomes in esophageal adenocarcinoma (EAC) patients. EAC originates from within the intestinal-type metaplasia, Barrett’s esophagus, a condition arising on a background of gastroesophageal reflux disease and associated inflammation.MethodsThis study used a druggable genome small interfering RNA (siRNA) screening library of 6022 siRNAs in conjunction with bioinformatics platforms, genomic studies of EAC tissues, somatic variation data of EAC from The Cancer Genome Atlas data of EAC, and pathologic and functional studies to define novel EAC-associated, and targetable, immune factors.ResultsBy using a druggable genome library we defined genes that sustain EAC cell growth, which included an unexpected immunologic signature. Integrating Cancer Genome Atlas data with druggable siRNA targets showed a striking concordance and an EAC-specific gene amplification event associated with 7 druggable targets co-encoded at Chr6p21.1. Over-representation of immune pathway–associated genes supporting cell growth included leukemia inhibitory factor, complement component 1, q subcomponent A chain (C1QA), and triggering receptor expressed on myeloid cells 2 (TREM2), which were validated further as targets sharing downstream signaling pathways through genomic and pathologic studies. Finally, targeting the triggering receptor expressed on myeloid cells 2-, C1q-, and leukemia inhibitory factor–activated signaling pathways (TYROBP–spleen tyrosine kinase and JAK-STAT3) with spleen tyrosine kinase and Janus-activated kinase inhibitor fostamatinib R788 triggered EAC cell death, growth arrest, and reduced tumor burden in NOD scid gamma mice.ConclusionsThese data highlight a subset of genes co-identified through siRNA targeting and genomic studies of expression and somatic variation, specifically highlighting the contribution that immune-related factors play in support of EAC development and suggesting their suitability as targets in the treatment of EAC.

Background: The purpose of this study was to reduce the length of stay of anterior cruciate ligament reconstruction patients within a private hospital in Ireland, reducing any non-value-added activity in the patient pathway, with the goal of increasing patient flow, bed capacity, and revenue generation within the hospital system, while maintaining patient satisfaction. Methods: We used a pre-/post-intervention design and Lean Six Sigma methods and tools to assess and improve the current process. Results: A reduction in inpatient length of stay by 57%, and a reduction in identified non-value-added activity by 88%, resulted in a new day-case surgery pathway for anterior cruciate ligament reconstruction patients. The pathway evidenced no re-admissions and demonstrated patient satisfaction. Conclusion: Six months post-project commencement, we had successfully achieved our goals of reducing our anterior cruciate ligament reconstruction patient’s length of stay. This study contributes to the growing body of published evidence which shows that adopting a Lean Six Sigma approach can be successfully employed to optimise care and surgical pathways in healthcare.

Background & Aims Esophageal adenocarcinoma (EAC) develops from within Barrett’s esophagus (BE) concomitant with gastroesophageal reflux disease (GERD). Wound healing processes and cellular transitions, such as epithelial–mesenchymal transitions, may contribute to the development of BE and the eventual migratory escape of metastatic cancer cells. Herein, we attempt to identify the genes underlying esophageal cellular transitions and their potential regulation by the low pH environments observed in GERD and commonly encountered by escaping cancer cells. Methods Small interfering RNA library screening and high-content imaging analysis outlined changes in BE high-grade dysplasia (HGD) and EAC cell morphologies after gene silencing. Gene expression microarray data and low pH exposures studies modeling GERD-associated pulses (pH 4.0, 10 min) and tumor microenvironments (pH 6.0, constant) were used. Results Statistical analysis of small interfering RNA screening data defined 207 genes (Z-score >2.0), in 12 distinct morphologic clusters, whose suppression significantly altered BE-HGD cell morphology. The most significant genes in this list included KIF11 , RRM2 , NUBP2 , P66BETA , DUX1 , UBE3A , ITGB8 , GAS1 , GPS1 , and PRC1 . Guided by gene expression microarray study data, both pulsatile and constant low pH exposures were observed to suppress the expression of GPS1 and RRM2 in a nonoverlapping temporal manner in both BE-HGD and EAC cells, with no changes observed in squamous esophageal cells. Functional studies uncovered that GPS1 and RRM2 contributed to amoeboid and mesenchymal cellular transitions, respectively, as characterized by differential rates of cell motility, pseudopodia formation, and altered expression of the mesenchymal markers vimentin and E-cadherin. Conclusions Collectively, we have shown that low pH microenvironments associated with GERD, and tumor invasive edges, can modulate the expression of genes that triggered esophageal cellular transitions potentially critical to colonization and invasion.

86 Background: The low survival for esophageal cancer is in part attributed to its high invasive potential and distant metastasis. In cancer, abnormal cell migration is an essential component of metastasis, and it is reasonable to consider that the conversion between different forms of morphology permits tumor cells to metastasise. Discovering regulators of esophageal cancer morphogenesis may aid in the development of novel targeted therapies that limit metastatic potential. Methods: GOhTRT cells were seeded and treated with siRNA (Human Druggable Genome, Dharmacon) by reverse transfection. Cells were fixed, immunostained for DNA, tubulin and actin and imaged with automated microscope. Cell images were processed using the InCell Analyzer software and the R statistical software systems CellHTS2 and RNAither. The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay. Results: 127 gene candidates greatly exhibited effects on F-actin and α-tubulin area staining. This list was refined to six high quality candidates (RRM2, ITGB8, GPS1, SPRY1, NOL1 and MYO9B). Silencing of GPS1, MYO9B and SPRY1 increased the rate of migration in a scratch wound assay, with 86.98% ± 3.097%, 75.78% ±5.454% and 72.97% ± 5.463% (p =0.0022) respectively. There was no significant difference in cell viability absorbance values for siGPS1 (0.9037 ± 0.06575; p =0.1905) and siSPRY1 (0.9088 ± 0.09849; p =0.2985), which suggests that the increased rate of wound closure previously seen is by virtue of migratory signalling as oppose to an increase in cell proliferation. Cell viability was decreased considerably in siRRM2 cells (0.2492 ± 0.02798; p <0.0001) and siMYO9B (0.4048 ± 0.04663; p<0.0001) in comparison to siNT cells (1.046 ± 0.07712). Conclusions: In summary, this screen successfully identified high confidence hits associated with cytoskeletal remodelling, some of which are already associated with metastasis in literature and database searches. Further mechanistic studies and gene pathway analysis of candidate genes will provide novel therapeutic targets which can be utilised to block the spread of cancer in patients.

70 Background: There is a pressing need to identify new therapeutic targets for esophageal adenocarcinoma (EAC). Hence, we utilized siRNA-screening libraries to identify genes impacting on EAC cancer cell growth to identify potential therapeutic targets. Methods: A “druggable genome” library (6,022 individual siRNAs) was utilized to examine EAC cell survival using the MTT assay. Statistical analysis combined the use of Z-factor, t-test and SSMD. EAC cell lines GohTRT, SKGT4 and OE33 were utilized. Functional validation of the resulting siRNAs utilized RT-PCR, Western Blot, ELISA and TOPFLASH assays. Results: siRNA library screening resulted in positive quality metrics (Z-factor>0.5) confirming its validity and further identifying 118 high confidence gene targets affecting EAC cell growth. Verification of these siRNA targets in multiple cell lines indicated a good level of concordance with the primary screening data. Bioinformatic and pathway mapping approaches of these targets emphasized links between EAC cell proliferation and regulators of inflammation and “immune cell processes” (LIF, C1Qa, C1r, C1s, GDF15, IL9R and TREM2). Pathological and transcriptomic studies demonstrated that LIF (FC=96.7; P<0.0001), GDF15 (EAC: FC=74; P<0.0001), C1Qa and TREM2 may be up-regulated in EAC biopsies. In functional work, exposure of EAC cell lines to recombinant or native proteins of C1q, LIF and GDF15 rescued the observed effects of their respective silencing in EAC cells (>90%,p0.001) and acted as potential growth promoters (>40%, p0.01). Auto-regulatory feedback loops were discovered in response to treatment with exogenous C1q and LIF in EAC cells. Signal transduction could be induced through b-catenin stabilization and STAT3 pathways in response to C1q and LIF treatment respectively. GDF15 was observed to act in a similar fashion to TGFb in scratch wound assays and additionally regulate Th17 type T-cell differentiation. Conclusions: Genes regulating EAC proliferation have been defined by siRNA library screening. We have identified secreted immune factors, not previously associated with EAC biology, capable of regulating EAC cell survival.