Metabolomic profiles throughout the continuum of colorectal carcinogenesis

The project "Metabolic profiles through the continuum of colorectal carcinogenesis" (MetaboCCC) was established as an answer to the ERA-NET translational cancer research (TRANSCAN) initiative, Joint Transnational Call 2012, where NFR and Kreftforeningen are partners. The MetaboCCC includes five partners/study centers 1) Division of Human nutrition, University of Wageningen, the Netherlands 2) International Agency for Research on Cancer (IARC), Lyon, France 3) German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), Heidelberg, Germany 4) Institute of Cancer Research, Medical University, Vienna, Austria 5) Bevital AS, Norway. Colon and rectal cancer are known to develop slowly through defined stages during a time-span of up to several decades. The stages are characterized by morphology, size, and invasiveness into surrounding tissue. A general feature of carcinogenesis is changes in the metabolism involved in energy production, cell-cell contact, and communication. The idea behind the project was to utilize new technologies that enables measurement of a large spectrum of metabolites in small quantities of blood plasma, and then use this information to characterize tumor stages. Laboratory analyses was performed using two divergent strategies: 1) Untargeted analysis quantifies up to several thousand signals/features for each subject in the study. The goal is to identify signals/features that discriminate between different stages of tumor development. A secondary goal is to identify the molecular nature of the best scoring features for further study. 2) Targeted analysis measures a predetermined set of biomarkers. This strategy incorporates defined internal standards for each metabolite to increase precision and facilitate reliable quantification of low-concentration metabolites which is likely to be important for the characterization of early stages of carcinogenesis. In short: The two analytical strategies would yield many (> 1000) metabolites/features with moderate (varying) precision, and fewer (hundreds) metabolites with high precision. The project aimed to measure metabolites, according to targeted and untargeted strategies, in 2300 blood samples across the spectrum from healthy controls through colorectal cancer stage 4 to identify biomarkers useful for prediction, staging, and prognosis. Because of difficulties concerning the recruitment of some patient categories, the final number of blood samples was 2030. Four partners (1,3,4, and 5) have contributed blood samples (plasma or serum) from ongoing or finished studies. Each study also collected other types of data, including age, sex, smoking, medication, co-morbidities, etc. The array of additional information was different between the studies with respect to nature, coding, stratification, name etc. Therefore, extensive data harmonization was undertaken in order to maximize the amount and utility of additional information/covariates across the studies. The measurement of targeted as well as a set of targeted biomarkers has been completed by partner 2 in Lyon, France. For the targeted analyses, a commercially available and validated kit for 186 metabolites (Biocrates AbsoluteIDQ p180 Kit) was used. This kit measures metabolites present at intermediate to high concentrations in blood such as acylcarnithines, amino acids, biogenic amines, and lipids. The Norwegian contribution of blood samples consisted of 400 serum samples from the finished NORCCAP study. In addition to targeted analysis at IARC. Bevital AS have also measured a panel of one-carbon metabolites, vitamins, amino acids, and protein markers for inflammation and kidney function - altogether 54 metabolites in the same samples. 26 of the metabolites overlapped with those measured at IARC. A comparison of analysis results demonstrated high reproducibility between the two analytical centers providing valuable feedback about the quality of analysis and validity of the study. Partner 5 (Bevital AS) has taken the main responsibility for the following main aim of the project: "Preventive or predisposing plasma metabolites discerning adenoma cases from controls using targeted metabolomics", this includes statistical analysis and scientific reporting in international scientific journals. Bevital AS will also similarly contribute to other main, and secondary aims as defined by the steering committee of the study.

Metabolomics is an innovative and powerful approach by which a large number of metabolites are systematically screened to characterize biological phenotypes with an unprecedented level of precision. New biomarkers that help characterize risk of progressio n along the pathway of colorectal carcinogenesis are urgently needed to tailor prevention strategies. By investigating plasma from individuals free of colorectal tumors, patients with colorectal adenoma, and patients with colorectal cancer (CRC, stage I-I V), the newly formed MetaboCCC Consortium aims to investigate changes in the metabolome along the continuum of colorectal carcinogenesis. We will perform metabolomic analyses in 2,300 plasma samples derived from well-defined populations of four TRANSCAN c ountries (the Netherlands, Germany, Austria, Norway) with assays completed at an expert site (France), using multiple discovery and replication sets to define biologic mechanisms of colorectal carcinogenesis. For a comprehensive and complementary strategy , we will apply both targeted and untargeted metabolomics. We aim to a) determine preventive or predisposing plasma metabolites discerning adenoma cases from controls, b) determine plasma metabolites that characterize CRC, compared with controls or adenom a cases, and c) test for markers that discern CRC stages. We expect that the discovery of novel metabolites in blood that define the transition between various stages of colorectal carcinogenesis can be used in the future for risk stratification, includi ng for tailored prevention strategies by endoscopy.