Abstract Background Despite recent advancements in the treatment of ulcerative colitis (UC), a substantial number of patients fail to achieve long-term remission. Persistent histological activity has been linked with poorer treatment outcomes. Histological remission is now an accepted treatment target; however there remains significant variability in the interpretation of UC histology. As such, there is a need for novel biomarker identification to aid assessment and ultimately predict disease relapse. Serum amyloid A (SAA) is an acute-phase protein, of which serum levels have shown promise as a biomarker in IBD1. This study aims to explore the utility of SAA levels in UC colonic tissue as a diagnostic biomarker for disease activity and progression. Methods Two cohorts were prospectively recruited, including healthy controls and UC patients. Sigmoid biopsies were collected and tissue explants generated. Tissue-conditioned media from these explants was collected and secreted SAA quantified using 54 V-plex ELISA. Demographic information, disease characteristics, endoscopic Mayo scores and disease progression were documented. Endoscopic remission was defined as a Mayo endoscopic sub-score ≤1. Disease progression was defined as the requirement for corticosteroid therapy, UC-related hospitalisation, UC-related surgery or the introduction of a new immunomodulatory agent in follow-up period. P values <0.05 were considered significant in analyses. Results The two cohorts included 11 healthy controls and 16 UC patients (endoscopic remission n=6). Active UC patients demonstrated significantly higher SAA concentrations than healthy controls (p=0.0013) and those in endoscopic remission (p=0.02). There was no significant difference in SAA concentrations between healthy controls and UC patients in remission (p=NS). UC patients in the lowest SAA concentration quartile had a significantly longer time to disease progression (p=0.0462) (Figure 1). Conclusion Quantification of SAA secretion in IBD ex-plants has potential as a biomarker of UC activity and progression. Further investigation of SAA as a biomarker in IBD is warranted. References 1. Chen R, Chen Q, Zheng J, Zeng Z, Chen M, Li L, et al. Serum amyloid protein A in inflammatory bowel disease: from bench to bedside. Cell Death Discov. 2023;9(1):154.

Background Immune checkpoint inhibitors (ICIs) reinvigorate anti-tumour immunity in oesophageal adenocarcinoma (OAC). However, emerging studies have identified novel immune-independent functions for immune checkpoints (ICs) in other solid tumour-types, whereby IC-intrinsic signalling in gastric cancer cells confers chemoresistance. This study explores immune-independent functions of ICs in OAC and if therapeutic blockade may enhance chemotherapy toxicity. Materials and Methods OAC cells were screened in vitro and in vivo (n=14 OAC human tissue biopsies) for a range of ICs (PD-1,TIGIT,TIM-3,LAG-3,A2aR,PD-L1,PD-L2,CD160) by flow cytometry. The phenotype of OAC cells expressing ICs was also assessed for features of stemness (ALDH, CD54), senescence (β-galactosidase) and invasiveness (vimentin) in the absence and presence of chemotherapy by flow cytometry. OAC cells were also treated with chemotherapy in the absence and presence of a MEK inhibitor to determine if MEK signalling regulated IC expression. Importantly, the effect of ICIs on the hallmarks of cancer in OAC cells was assessed which included: OAC cell viability (CCK-8 assay and western blot to assess Bcl-xL and Bcl-2 levels), proliferation (BrdU assay and ki67 expression by intracellular flow cytometry), chemo-sensitivity (annexin-V propidium iodide assay and cell cycle analysis by flow cytometry and expression of chemotherapy efflux and influx pumps by western blot: ATP7a, ATP7b, CTR1 and ABCB9), metabolism (seahorse), invasiveness and stemness characteristics (vimentin and aldefluor assay, respectively by flow cytometry) and DNA repair (γH2ax by flow cytometry to assess levels of DNA repair and the expression of DNA repair genes were quantified by qPCR: MLH1, SMUG1, PARP1, MMS19) was assessed in OAC cells. Results A subpopulation of stem-like, senescent and vimentin+ OAC cells were enriched for ICs, which was enhanced by FLOT and CROSS chemotherapy regimens. IC expression increased on the surface of OAC cells 48h post-chemotherapy treatment and was sustained up to 3 weeks post-treatment in vitro. Inhibition of pro-survivla MEK signalling reduced chemotherapy-induced upregulation of ICs. Blockade of PD-1, TIGIT, A2aR, TIM-3 and PD-L1 decreased proliferation, DNA repair, induced apoptosis and enhanced toxicity of FLOT in OAC cells. Blockade of TIGIT decreased pro-survival Bcl-xL factor, induced cell death and promoted a more glycolytic phenotype in OAC cells. Conclusions Several novel ICs have been identified as potential targets to enhance chemotherpay efficacy in OAC. Upregulation of ICs on OAC cells following chemotherapy may represent potential mechanisms of chemo-immune resistance for stem-like, senescent and vimentin+ aggressive cancer cell clones. Combining ICIs with chemotherapy may synergise with chemotherpay in OAC patients via immune-independent mechansims and boost response rates to current standards of care. Further studies are warranted through clincal trials to further establish synergistic ICI-chemotherapy combinations in OAC. Disclosure Information M. davern: None. C. Buckley: None. C. Fitzgerald: None. A.B. Heeran: None. N.E. Donlon: None. J. McGrath: None. R. O' Brien: None. F. O' Connell: None. B. Murphy: None. N. Lynam-Lennon: None. J.V. Reynolds: None. S.G. Maher: None. A.D. Sheppard: None. A. Bhardwaj: None. A. Bhardwaj: None. C. Butler: None. N. Ravi: None. J. Lysaght: None.

Esophageal adenocarcinoma displays a poor prognosis and current treatments are often not curative. Pathological TNM-stage is a prognostic parameter, but a better understanding of the pathophysiology of esophageal adenocarcinoma is needed to better predict survival. Recent work in other malignancies indicated an important role for the regulator microRNA-126 (miR-126) in tumors. The aim of this study was to investigate the function of miR-126 in esophageal adenocarcinoma and to correlate expression of miR-126 with tumor cell behavior and patient survival. Functional assays were performed in esophageal adenocarcinoma cell lines (OE33) in vitro by overexpressing or antagonizing miR-126 and assessing cellular processes linked to the hallmarks of cancer. In vivo pre-treatment biopsies of 58 patients with esophageal adenocarcinoma who underwent neoadjuvant chemoradiotherapy and surgery were analyzed for miR-126 expression in tumor cells by qRT-PCR and patient survival was analyzed by Kaplan–Meier and Cox regression. In OE33 cancer cells, stable overexpression of miR-126 modest though significantly altered expression of genes related to cell death (MEK1) and DNA repair (POLB and TERF1) was observed. Also the secretion of the angiogenic and pro-inflammatory factors, VEGF, IL-1β, and IL-6 were regulated by miR-126 ( P < 0.029). Importantly, miR-126 was found to be a regulator of cell viability in OE33 cells. Overexpressing ( P = 0.043) and antagonizing ( P = 0.035) miR-126 showed reciprocal effects on tumor cell viability and significantly regulated expression of pro- and anti-apoptotic genes, TP53, and GATA6 ( P < 0.031). In patients, high levels of miR-126 expression in pre-treatment tumors were significantly associated with poor survival ( P = 0.031). In multivariable analysis, high miR-126 ( P = 0.038) together with ypN-stage ( P = 0.048) were shown to be independent risk factors for poor survival. In conclusion, high expression of miR-126 in esophageal adenocarcinoma prevents tumor-cell death and is associated with poor patient survival. This study warrants further analysis of miR-126 as biomarker or potential therapeutic target for OAC. Impact statement Esophageal adenocarcinoma is a common form of cancer of the esophagus. It has an increasing health impact as it is associated with very poor patient survival. A better understanding of the pathophysiology of this cancer is needed to identify better treatment strategies and to provide a better prognosis for these patients. MicroRNAs have emerged as important molecular regulators of cancer cell viability and proliferation. The aim of our study was to investigate the role of one very well established microRNA, miR-126, in esophageal adenocarcinoma. Our research shows clear experimental evidence that miR-126 controls cell viability of esophageal adenocarcinoma cells. High (over)expression of miR-126 increased the viability of these cells. Our preclinical data were shown to be clinically relevant for this field of oncology. In an independent validation study of esophageal adenocarcinoma biopsies, we confirmed that high miR-126 expression in tumor cells was an independent risk factor for poor patient survival.

Introduction Oesophageal adenocarcinoma (OAC) and rectal adenocarcinoma are treated with neoadjuvant chemoradiotherapy in order to reduce tumour size prior to surgery however only 10%–30% of patients have a complete pathological response. Inflammatory and angiogenic mediators in the tumour microenvironment (TME) have many functions, such as enabling evasion of anti-tumour immune responses by disabling infiltrating dendritic cells (DCs) and have been linked with radioresistance. Tumour Conditioned Media (TCM) from colonic cancer has been shown to strongly inhibit DC maturation. Our aim was to understand if this DC inhibition extends to other cancers of the gastrointestinal tract, to investigate if radiotherapy influences this and to profile constituents of TCM that may influence DC maturation. Material and methods TCM from 0Gy or 2Gy-irradiated cell lines or tumour biopsy explants, was used to pre-treat monocyte-derived DCs prior to stimulation with LPS to measure DC maturation based on DC cell surface markers (HLA-DR, CD86, CD54, CD80, CD83 and PD-L1) and two cytokine levels (IL12 p70 and TNF alpha). Inflammatory and angiogenic mediator multiplex ELISAs were used to profile the TCM of oesophageal and rectal adenocarcinoma. Results and discussions DCs remained responsive to LPS following pre-treatment with OAC cell line TCM, whereas extensive inhibition was induced by CRC cell line TCM. ex vivo TCM from different gastrointestinal adenocarinoma types induced different effects on DC maturation with oesophageal inducing DC activation, rectal inducing minor activation and colonic inducing inhibition of DC maturation markers. Interestingly, all cancer types induced DC inhibition of secreted TNF alpha. It was also found that 2Gy-irradiated TME induced significant inhibition of DC maturation for irradiated rectal adenocarcinoma and no effect with irradiated oesophageal cancer. Differential levels of inflammatory (IL2) and angiogenic mediators (Ang2 and bFGF) in TCM of GI tumours correlated with DC maturation. Conclusion Overall, this study offers new evidence that there are differences in the human TME from different gastrointestinal (GI) cancers which can directly induce varying levels of inhibition of LPS-induced DC maturation markers, whilst all inhibit secreted TNF alpha.

The risk of recurrence following radiation therapy remains high for a significant number of prostate cancer patients. The development of in vitro isogenic models of radioresistance through exposure to fractionated radiation is an increasingly used approach to investigate the mechanisms of radioresistance in cancer cells and help guide improvements in radiotherapy standards. We treated 22Rv1 prostate cancer cells with fractionated 2 Gy radiation to a cumulative total dose of 60 Gy. This process selected for 22Rv1-cells with increased clonogenic survival following subsequent radiation exposure but increased sensitivity to Docetaxel. This RR-22Rv1 cell line was enriched in S-phase cells, less susceptible to DNA damage, radiation-induced apoptosis and acquired enhanced migration potential, when compared to wild type and aged matched control 22Rv1 cells. The selection of radioresistant cancer cells during fractionated radiation therapy may have implications in the development and administration of future targeted therapy in conjunction with radiation therapy.

Oesophageal adenocarcinoma is an exemplar model of obesity-associated cancer. Locally advanced disease is treated with neoadjuvant chemoradiotherapy, and survival rates are highest in patients demonstrating a pathological response following neoadjuvant therapy. Given that 55 % of oesophageal adenocarcinoma patients are obese, uncovering the effect of adipose tissue on radioresponse is clinically relevant. This study investigates if adipose tissue activates genomic instability events in radioresponsive (OE33P) and radioresistant (OE33R) oesophageal cancer cell lines and tumour samples.

Contemporary clinical management of Barrett's oesophagus has highlighted the lack of accurate predictive markers of disease progression to oesophageal cancer. This study aims to examine alterations in mitochondrial energy metabolism profiles across the entire disease progression sequence in Barrett's oesophagus. An in-vitro model was used to screen 84 genes associated with mitochondrial energy metabolism. Three energy metabolism genes (ATP12A, COX4I2, COX8C) were significantly altered across the in-vitro Barrett's disease sequence. In-vivo validations across the Barrett's sequence demonstrated differential expression of these genes. Tissue microarrays demonstrated significant alterations in both epithelial and stromal oxidative phosphorylation (ATP5B and Hsp60) and glycolytic (PKM2 and GAPDH) protein markers across the in-vivo Barrett's sequence. Levels of ATP5B in sequential follow up surveillance biopsy material segregated Barrett's non progressors and progressors to HGD and cancer. Utilising the Seahorse XF24 flux analyser, in-vitro Barrett's and adenocarcinoma cells exhibited altered levels of various oxidative parameters. We show for the first time that mitochondrial energy metabolism is differentially altered across the metaplasia-dysplasia-adenocarcinoma sequence and that oxidative phosphorylation profiles have predictive value in segregating Barrett's non progressors and progressors to adenocarcinoma.

Introduction Deoxycholic acid (DCA), a component of bile, has been associated with the development and progression of Barrett9s oesophagus (BO). In addition, the “mutator-phenotype hypothesis” states benign cells with high rates of random mutations have an inherent predisposition for malignant conversion. Mitochondria are highly susceptible to random mutations due to inefficient DNA repair mechanisms. The role of mitochondrial mutations and dysfunction in Barrett9s progression is unknown. The aim of this study was to investigate the levels of random mitochondrial mutations (RMMs) and dysfunction in BO and determine if DCA drives progression. In parallel, determine if a hydroxylase inhibitor, dimethyloxalylglycine (DMOG), could rescue these effects. Methods We assessed mitochondrial dysfunction and mutagenesis using different models in vitro, ex vivo and in vivo. RMMs in BO extending to oesophageal adenocarcinoma (OAC) were examined. Mitochondrial function (reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondrial mass and cytochrome c) +/− DCA +/− DMOG were measured. A large scale gene array assessing oxidative stress and antioxidant defence was used to determine potential genes DMOG therapy may target. Results In vitro; RMMs were higher in Qh (intestinal metaplasia [IM] cells) compared with GO (high grade dysplasia [HGD] cells) (p=0.06) and OE33 (OAC cells) (p=0.01). DCA induced mitochondrial dysfunction at all stages of disease progression (p<0.04). DCA increased mutagensis in Qh cell lines only. Using ex vivo explants, cytochrome c release was significantly increased in Barrett9s compared to matched normal controls (p=0.0006). DCA significantly decreased cytochrome c secretion in Barrett9s patients (p=0.015). In vivo; two distinct Barrett9s IM groups were evident; those with low RMMs and a small cohort with significantly higher levels of RMMs (p<0.005). DMOG caused a significant reduction in mitochondrial dysfunction in Go and OE33 cells (p<0.05). DMOG down-regulated 3 genes, CYGB, FOXM1 and GLRX2 (13, 6 and 4 fold respectively), validations of these are being performed. Conclusion Mitochondrial instability appears to be an early event in BO. DCA significantly alters mitochondrial function. Two distinct Barrett9s patient groups are evident. Applying the “mutator-phenotype hypothesis”; random mitochondrial mutation may act as a potential biomarker in differentiating IM patients into low and high risk groups for malignant conversion. DMOG may have the potential to reduce this mitochondrial instability in Barrett9s oesophagus. Competing interests None declared.

Our results demonstrated that the methylation of HIST1H4K most likely increases with the age.We do not observe any methylation among the young asymptotic males, while the frequency of HIST1H4K methylation did not differ between the PC patients and BPH controls.The promoter hypermethylation of HIST1H4K shows weak correlation with high Gleason score, bit in order to clarify its role for the development of aggressive PC, further investigation in enlarged sample of patients and BPH controls is needed.In our study HIST1H4K did not show correlation with PSA and the tumour stage.The contribution of this gene for development of metastases needs further investigation.Conclusions: The promoter hypermethylation of HIST1H4K is most likely associated with the appeared with age changes in the prostatic gland and shows weak correlation with high Gleason score.Further investigations are needed to confirm this observation.Promoter hypermethylation of RASSF2 was not found in the studied cohort of PC patients and controls.This marker probably will not be suitable for PC diagnostic testing in Bulgaria.

Introduction Obesity is strongly associated with oesophageal adenocarcinoma (OAC). However, its role in regulating radiosensitivity and genomic instability is unknown. We developed an isogenic model of radioresistance in OAC called OE33R. We assessed levels of anaphase bridges, a functional genomic instability event, in OE33R compared to an age and passage matched control (OE33P). Spindle assembly checkpoint (SAC) control genes involved in regulating genomic instability were analysed in both cell lines following exposure to adipose conditioned media (ACM). Radiosensitivity following ACM treatment was investigated in OE33R and OE33P cell lines. The aim of this study was to characterise the isogenic radioresistant OAC cell model in terms of genomic instability, SAC gene expression and survival in response to adipose conditioned media (ACM) cultured from obese and nonobese OAC patients. Methods OE33R and OE33P cell lines were cultured with ACM from obese and nonobese patients. We assessed anaphase bridges in both lines and quantified the number of bridges present over the total cell number. Expression of five SAC genes (MAD2L2, BUB1B, CDC20, CENPE, and ESPL1) was assessed using qPCR. Survival was determined in both cells lines following ACM treatment using a clonogenic assay. Results OE33P and OE33P showed a significant increase in anaphase bridges in response to ACM (p<0.05, p<0.001 respectively). This increase in anaphase bridge formation was three times greater in the resistant line (p<0.05). Levels of anaphase bridge formation correlated with obesity status (BMI and VFA, p<0.05). OE33R cells treated with ACM showed significantly increased expression of the SAC genes MAD2L2 and BUB1B compared to controls (p<0.01). Expression of MAD2L2 and BUB1B correlated with obesity status (p<0.05). OE33P cells treated with ACM showed increased radioresistance (p<0.05). In contrast, in the resistant OE33R cell line, ACM treatment reversed this radioresistance (p<0.001). Conclusion Obesity drives genomic instability and alterations in SAC gene expression in radioresistant OAC and alters radiosensitivity in OAC. Competing interests None declared.