Research
Biomarkers discovery for patient surveillance and early diagnosis of sporadic Colorectal Cancer
This project stared in 2012 and is a collaborative project between the team of Prof. Denis Franchimont at the Erasme hospital (ULB), the pathology unit of CHU Liège, the laboratory of mass spectrometry (CART), the GIGA platforms, the BHUL and our team at ULiège. The plan cancer funded this project (2012-2015), which also received support from the TELEVIE (2015-2017).
Proteomics biomarkers discovery for the early detection of colorectal cancer was our initial general aim. We focused on early diagnosis by studying the proteome of FFPE residual samples. Our strategy involved also the study of precancerous lesions as these are at higher risk of adenocarcinoma transformation. Hence, in this study, we included adenomas (low grade dysplasia, high grade dysplasia and sessile serrated adenoma). The detection of the last two categories of adenomas is of major interest in clinics, as these are known to be prone to shorter term ADK transformation than low grade classical adenomas. In this context we also focused on sessile serrated lesion (SSL) and their discrimination from other malignant and non-malignant lesions as hyperplastic polyps (HP). Some specific and sensitive tissue markers could be used during the pathological diagnosis or as specific tracer during colonoscopy which is the current gold standard method for CRC surveillance and screening. Some protein markers identified in SSL might also explain the different sequence of transformation associated with these specific more aggressive lesions.
Surveillance and diagnosis of colorectal cancer in IBD patients
The colitis associated cancer (CAC) is specifically associated with inflammatory bowel disease. Since several years we also focus on this specific complication of IBD. Dysplasia and neoplasia associated to inflammation occur in zones affected by chronic inflammation and recurrent tissue remodeling. Such dysplasia is at higher risk of ADK transformation and CAC progression and shows a different sequence of malignant transformation from sporadic ones.
Diagnosis during examination by colonoscopy is challenging as lesions are often flat, depressed or found within an inflammatory area. Moreover, the pathological examination of the resected tissue (polypoid or not) is rather complicated as remodeling in the epithelial tissue and cell proliferation are common features observed with inflamed mucosa or cells in dysplasia. In this context, our project involved the comparison of the proteome of epithelial cells isolated in the dysplastic zones of FFPE tissues originated from IBD patients (UC and CD dysplasia), of epithelial cells selected within zones with inflammation and selected on normal paired tissue. We identified a tissue protein named SLC12A2 (NKCC1), which is able to discriminate with high sensitivity and specificity, colorectal lesion (from low grade adenoma to ADK stage: CAC or CRC) from pure inflammatory or normal tissues in IBD and the general population (Merli M A et al, JCC 2021). Currently we are studying NKCC1’s role and fate during colorectal carcinogenesis, using a functional approach through the use of colorectal cancer cell lines and more importantly, human intestinal organoids and tumoroids derived from IBD and CRC. This project involves a close collaboration with CHU surgery department (C Coimbra and M Decker) pathology department (Prof P Delvenne, N Blétard, J Somja) and the biothèque of our institution (BHUL). This project was funded through several Télévie PhD fellowships (past Angela-Marie Merli and currently Pierre ADAM), the Fondation Leon Frédéricq and Uliege.
IMIDS and IBD
IMIDs group several pathologies affecting the digestive tract, joints and skin and sharing common inflammatory and immune features. Inflammatory Bowel Diseases (IBDs) include Ulcerative Colitis (UC) and Crohn Disease (CD) which are complex and multifactorial digestive syndromes presenting similar clinical symptoms. However, their treatments and managements differ and biomarkers that could improve patient diagnosis, prognosis and follow-up are needed.
Relapse prediction in CD
We started in 2012 a program (funded by an ECCO grant) for the discovery of biomarkers for the prediction of relapse in patient showing longstanding remission induced by anti-TNF therapy and stopping treatment, using the STORI trial patient samples. We addressed this challenge using discovery and targeted proteomics via our collaboration with The development of a specific targeted method (Selected reaction monitoring, SRM) for the validation of potential biomarkers has been developped during the FEDER action portofolio 2017-2023 and the PREDIMID project, managed by Nicolas PIERRE. The collection of samples for the final validation of the selected biomarkers using the developped method has been performed thanks to the samples of the SPARE clinical trial. This clinical trial was one component of the European funded Horizon 2020 BIOCYCLE program, headed by Edouard LOUIS which focused on many clinical aspects and biomarker research for relapse prediction (2015-2022) (LOUIS E. et al. 2023 Lancet. Gastroenterology and Hepatology).
In the STORI cohort, we also applied the proximity extension assay (PEA) technology (Olink) to measure serum proteins (Pierre N et al, GUT, 2020 and Pierre et al, GUT 2022). Overall, both proteomics results showed that, after stopping the anti-TNFα, short-term relapsers (< 6 months) and mid/long-terme relapsers (> 6 months) were associated with distinct blood protein profiles including proteins associated with inflammation and acute phase reactants and proteins involved in immunity. The biological state of mid/long-term relapsers preceding the inflammatory flare (short-term relapsers), our results might gain insight into the dynamics of the biological events occurring before relapse. Part of these results using both proteomic technologies were confirmed within the SPARE samples (ongoing). Based on these evidences, we proposed a new definition of a novel treatment target defined as the “biological remission” and will pursue the confirmation of these results and potential biomarkers for relapse prediction, thanks to an ECCO Grant 2022 (N Pierre) and a third GETAID: Groupe d'Étude Thérapeutique des Affections Inflammatoires du Tube Digestif (GETAID) cohort named CREDO which evaluates the relapse of Crohn’s disease patients under treatment.
Mucosal healing biomarkers in Crohn’s disease
Another clinical question addressed within the IBD projects of our laboratory is the discovery of biomarkers associated with mucosal healing. Thanks to new therapeutic developments in IBD, mucosal healing has become a key treatment objective beside clinical remission. The PREDIMID project also addressed this specific question using proteomics on biopsies and blood samples collected in the daily clinical practice in the university Hospital (CHU de Liège). A discovery proteomic study was conducted on ulcer edges of CD patients and allowed to screen ileal and colonic proteins to highlight distinct profiles associated with these lesions specifically in the ileum and the colon (Pierre et al, JCC 2019). SRM and PEA were then used to identify circulating (serum) biomarker candidates for the non-invasive monitoring of ulcers in the ileum and in the colon of CD patients. The different pathophysiology driving ileal and colonic CD is also one of our research focusses (Pierre N et al, Alimentary Pharmacology & Therapeutics, review, 2021).
Intesinal fibrosis in IBD
A major complication of IBD is fibrosis. Tissue healing after cumulative tissue damages occurring during uncontrolled patient flares and relapses may lead to tissue remodeling and collagen fibers deposition within and under the mucosa and muscles. This phenomenon may impair bowel function, motility in CD and UC and can cause bowel strictures in CD. By comparing the proteome of epithelial cells isolated within CD tissue sections (Formalin fixed paraffin embedded sample) showing different stages of fibrosis and inflammation, we highlighted ER stress and AGR2 as specific process and proteins associated with CD fibrosis. Functional characterization of AGR2 role on fibrogenesis has been shown using the intestinal model of Fibroblast to myofibroblast transition (FMT) and epithelial cell line models (Vieujean et al, 2021, JCC). This work was funded by FNRS (CSD project of Sophie Vieujean) and the CHU (FIRS) as well as Uliege.
We have characterised ER stress by tissue evaluation of different ER stress chaperones associated with fibrosis (as AGR2) in adults and paediatric CD fibroinflammatory stenotic tissues, in the colon and the ileum (PhD of Emeline Bequet) supported by the the Fondation Leon Frédéricq (Elvira Eyckelberg grant), CHU of Liège and Uliège.
The investigation of the role of the epithelium, in the fibrosis initiation and progression, is ongoing. We are specifically addressing the response of epithelial cells to ER stress in Ulcerative colitis, where intestinal fibrosis features are present in the mucosae and below the muscularis mucosae. We are using intestinal organoids of UC and healthy individuals to identify the response of intestinal epithelial cells to ER stress exposure and downstream molecular changes induced. The secreted mediators capable of inducing FMT in response to ER stress induction, as extracellular AGR2, are under investigation. This project is funded by the FRIA (PhD- fellowship of Marty STEPNIAK), the Fondation Leon Frédéricq and Uliege.
We are also involved in the Aspire project (Pfizer research project grant 2020) in collaboration with the INFINITE team of university of Lille, Prof L. Dubuquoy, Prof D. Launay and Dr Silvia Speca. Title and focus: Long term 3D inverted organoids model of UC-associated intestinal fibrosis: a new tool for investigating relevant molecular signatures of response to Tofacitinib.
Mucosal barrier integrity and Homeostasis loss
Intestinal homeostasis loss is present in IBD under flare and also described for other systemic chronic and inflammatory pathologies. We study this primary moment in disease development through different projects. We addressed the proteomic changes associated with CD, UC in remission and also healthy individuals. Indeed, these subjects and patients may show perturbation of the mucosal barrier and Leaky gut (monitored by the presence of fluorescein leaks during Laser confocal micro-endomicroscopy examination during colonoscopy thanks to the Cellvizio® probe system). The defect of the epithelial barrier integrity associated with IBD and not with healthy subject might be involved in homeostasis loss that IBD patients show constitutively. This CHU project (FIRS), also funded by the Fondation Leon Frédéricq is done in collaboration with J-P Loly (CHU of Liège) (Loly, J.-P et al, IBD, 2023)
The results obtained highlight new protein targets associated with IBD induced epithelial homeostasis loss which are now investigated within an EOS program (HOMISTASIS - Host-microbe interaction in intestinal homeostasis: unravelling the mechanisms involved in the onset of multiple inflammation-related diseases, coordinator P Cani, UCL Call "EOS - Excellence of Science" 2021 | TODO / TODO (frs-fnrs.be)).
