University of Dundee Novel biomarkers for risk stratification of Barrett's oesophagus associated neoplastic progression-epithelial HMGB1 expression and stromal lymphocytic phenotype Porter, Ross J.; Murray, Graeme I.; Brice, Daniel P.; Petty, Russell D.; McLean, Mairi H. Published in: British Journal of Cancer DOI: 10.1038/s41416-019-0685-1 Publication date: 2020 Document Version Peer reviewed version Link to publication in Discovery Research Portal Citation for published version (APA): Porter, R. J., Murray, G. I., Brice, D. P., Petty, R. D., & McLean, M. H. (2020). Novel biomarkers for risk stratification of Barrett's oesophagus associated neoplastic progression-epithelial HMGB1 expression and stromal lymphocytic phenotype. 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Sep. 2021 1 Novel biomarkers for risk stratification of Barrett’s oesophagus associated neoplastic 2 progression - epithelial HMGB1 expression and stromal lymphocytic phenotype 3 4 Running title – Novel biomarkers in Barrett’s oesophagus 5 6 Ross J Porter1, Graeme I Murray1, Daniel P Brice1, Russell D Petty2, Mairi H McLean1 7 8 1School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Foresterhill, 9 Aberdeen AB25 2ZD 10 2Division of Molecular and Clinical Medicine, School of Medicine, University of Dundee, 11 Dundee DD1 1GZ 12 13 Corresponding Author: 14 Dr. Mairi H McLean 15 Institute of Medical Sciences 16 School of Medicine, Medical Sciences and Nutrition 17 University of Aberdeen 18 Aberdeen AB25 2ZD 19 20 e-mail: [email protected] 21 22 Keywords; Barrett’s oesophagus, oesophageal cancer, HMGB1, lymphocytes, biomarker, 23 epithelial cell 24 1 25 ABSTRACT 26 Background: The incidence of oesophageal adenocarcinoma is increasing globally. Barrett’s 27 oesophagus (BO) is a pre-malignant condition with no biomarker to risk stratify those at 28 highest risk of dysplasia and malignant transformation. Methods: Subcellular epithelial 29 protein (HMGB1, p53, RUNX3) expression, alongside expression of CD20, CD4, CD8 and Foxp3 30 to characterise stromal B lymphocyte, and helper, cytotoxic and regulatory T-lymphocyte cell 31 infiltrate, respectively, was assessed by immunohistochemistry in 218 human tissue samples 32 including normal oesophageal/gastric biopsies (n=39), BO (non-dysplasia, dysplasia, non- 33 dysplastic background from progressors to dysplasia or cancer, n=121), and oesophageal 34 adenocarcinoma (n=58). Results: There is a dynamic subcellular epithelial expression of 35 HMGB1 (loss of nuclear, emergence of cytoplasmic), associated with epithelial p53 expression 36 and differential immune cell phenotype in oesophageal neoplastic progression. We identify a 37 protein signature and lymphocyte infiltrate in non-dysplastic BO when progressive disease 38 (dysplasia or adenocarcinoma) is present but not histologically represented in the biopsied 39 field. There is a dynamic stromal lymphocytic infiltrate in oesophageal neoplastic progression. 40 Conclusions: This data reveals novel insights into the microenvironment of BO and 41 progression towards cancer, and identifies a novel high risk biomarker of disease progression 42 to aid surveillance strategies to identify early progression and impact future incidence of 43 oesophageal cancer. 44 45 46 2 47 BACKGROUND 48 The global incidence of oesophageal cancer is increasing with over half a million cases 49 worldwide in 2018, accounting for 1 in 20 of all cancer deaths. In the UK,1 there are 9000 new 50 cases per year with a 15% 5 year survival.2 Barrett’s oesophagus (BO) is a pre-malignant 51 condition for oesophageal adenocarcinoma that affects 1.6-8% of the UK population.3 BO is 52 defined by histological evidence of epithelial metaplasia from normal stratified squamous 53 epithelium to mucin-secreting columnar epithelium in the distal oesophagus. These 54 metaplastic cells can undergo further transformation to dysplasia or malignancy. Although 55 the lifetime risk of transformation remains under debate, a previous UK study of nearly 8000 56 patients suggested this could be as high as 1 in 14 patients, although a risk of approximately 57 0.5% per annum has been more widely reported.4 The annual incidence of oesophageal 58 adenocarcinoma is 0.33% and 1.40% in non-dysplastic and dysplastic BO, respectfully.5,6 59 Cancer incidence has been estimated to be 40-times greater in high grade dysplasia, 60 compared to the general population.7 Currently, it is not possible to predict those with BO at 61 high risk of progression to dysplasia and malignancy. Therefore, all patients are offered 62 interval endoscopic surveillance with biopsy to detect cell dysplasia or early cancer amenable 63 to endoscopic therapy.8 This strategy carries significant resource implications and subjects 64 individuals to repeated invasive investigation. There is clearly a clinical need to identify those 65 at highest risk of progression at the point of diagnosis and to focus endoscopic and clinical 66 resource accordingly. Currently, the pathogenesis of metaplastic-dysplastic-malignant 67 progression is not fully understood.3,9 However, there have been a variety of proposed 68 biomarkers to aid this risk stratification10, including transcriptional changes,11 tissue 69 microRNAs12,13 or circulating glycoproteins14 and breath volatiles.15 3 70 Identifying novel cellular mechanisms that underlie BO pathogenesis and progression 71 would identify biomarkers of risk, inform less invasive and more sensitive monitoring 72 strategies and by detecting progressive disease early, impact incidence and prognosis of 73 oesophageal cancer. 74 With this clinical problem in mind, our aim was to define the role of the protein high 75 mobility group box-1 (HMGB1) in BO and progression to cancer. Due to its cellular functions, 76 and link to key protein targets, we hypothesized that HMGB1 is important in the pathogenesis 77 of BO to cancer. HMGB1 is a ubiquitous nuclear protein that binds to the minor grove of DNA 78 to stabilise the genome and regulate gene expression.16 Cellular stress results in 79 phosphorylation of HMGB1, inducing cytoplasmic and extracellular shuttling.17 Once 80 extracellular, HMGB1 influences epithelial cell behaviour and immune cell responses.18,19 81 HMGB1 is differentially expressed in malignancy at a number of body sites, including liver, 82 stomach, colon, bladder, pancreas, prostate and cervix. In squamous oesophageal cancer, 83 HMGB1 promotes lymphangiogenesis by regulating expression of VEGF-C and VEGF-D, and is 84 negatively correlated to survival.20 There is no data on expression of HMGB1 in BO or 85 oesophageal adenocarcinoma. 86 We propose that identifying important effector proteins, downstream of HMGB1, is 87 key to fully characterising the role of HMGB1 in pre-malignant and malignant pathologies. 88 p53 is a pivotal tumour suppressor protein that becomes dysregulated in various cancers.21 89 In oesophageal adenocarcinoma, approximately 75% of patients have p53 mutations and 90 consequently express strong nuclear p53.22 In BO, only foci of dysplasia exhibit p53. Some 91 centres incorporate p53 expression as a diagnostic aid for identifying dysplasia.8 Of interest 92 here, HMGB1 can facilitate p53-DNA binding, induce a p53-dependent senescent growth 93 arrest, and complex with p53 to mediate autophagy and apoptosis; localisation and 4 94 expression of each protein influences the other.23–25 We hypothesise that loss of nuclear 95 HMGB1 expression could impact p53 expression in oesophageal neoplastic progression. 96 Similarly, runt-related transcription factor 3 (RUNX3) is a highly-conserved 97 transcription factor important in the activation, proliferation and differentiation of 98 lymphocytes.26 Promoter hypermethylation of RUNX3 is associated with an increased risk of 99 progression, as well as poorer survival in oesophageal cancer.27 Hypermethylation of RUNX3 100 occurs as an early event in BO and is an independent risk factor for progression to dysplasia 28 101 or oesophageal adenocarcinoma. HMGB1 has been identified as a potent inducer of an 102 interferon-γ producing TH17 lymphocyte immune cell response, via regulation of transcription 103 factors T-bet and RUNX3, leading to progression of atherosclerosis.29 Therefore, we 104 hypothesise that RUNX3 may be an important downstream mediator influenced by HMGB1 105 expression. 106 HMGB1 also co-ordinates immune cell function, although this is not well 107 characterised.30 The mechanisms by which extracellular HMGB1 influences immune activity 108 is complex and dependant on post-translation modification status through direct interaction 109 with transmembrane receptors such as RAGE or