ABSTRACT: Interventions: Group 1: Patients diagnosed with colorectal cancer aged 18-49 years
Group 2: Comparators without colorectal cancer aged 18-49 years
Primary outcome(s): Specific goals of the individual sub-projects of the OUTLIVE study
Sub-project 3
This sub-project (SP) aims to decipher mechanisms of diet-related metabolic product (metabolite) patterns and their influence on biological aging processes in the context of colorectal carcinogenesis. The data from the metabolome analyzes will be combined with microbiome, epigenome, genome and proteome data from the same analyzed samples from the other sub-projects (TP4-6) to generate a meta-dataset (TP7). This data set should enable a prediction of the biological age and the risk of recurrence in chronologically young CRC patients in an automated test (TP8). Together with the results from TP9, the question should be answered whether the difference between chronological and biological age is driven by nutrition and can be prevented by personalized nutritional intervention in order to avoid relapses, especially in chronologically young CRC patients. To investigate the functional consequences of diet-related metabolic conditions on tumor development in the colon, AP1 will generate intestinal 3D organoids from colon biopsies obtained from non-CRC patients (irritable bowel syndrome, celiac disease, food hypersensitivity, etc.). In addition, the body composition of the examined patients is monitored by a bio-impedance analysis before the endoscopy. The long-term observation and diet type determination of the metabolism of the non-CRC patients examined is carried out as part of TP9 in close cooperation with Perfood GmbH. The 3D human colon organoids are then cultured under different metabolic conditions. Malignant transformation of 3D organoids is analyzed by examining proliferation, differentiation and metabolism using real-time cell analysis, fluorescence microscopy, immunohistochemistry, Western blot, qPCR and flow cytometry. In AP2, serum samples from CRC (TP1) or non-CRC patients (AP1) will be used for NMR-based metabolome analysis. The metabolome data of the non-CRC patients are supplemented by clinical parameters (blood sugar, insulin, HbA1c, vitamins, CRP, leukocyte count, etc.), which are determined in TP9 before the endoscopy. At the same time, stool samples are collected to examine the intestinal inflammation (faecal blood, calprotectin, C3 and IgA). These data will be correlated with the composition of the faecal microbiome (TP4).
Sub-project 4
The aim of TP4 is to identify new epigenomic and microbiomic markers for the earlier detection of the CRC in plasma and stool samples from individuals in a defined cohort of subjects (TP1, TP3). Here, the methylation pattern of cfDNA samples from young CRC patients and cfDNA samples from non-CRC patients (TP3 & TP9) will be recorded. In parallel, the composition of the microbiome in stool samples from the same individuals will be determined using 16S rRNA sequencing. The markers obtained are to be prioritized by bioinformatic analyzes in cooperation with the partners in TP3, TP5, TP6 and TP7 with regard to their diagnostic potential and included in the Multimarker Panel (TP8).TP4 is divided into three work packages (AP). In WP1, enrichment-based sequencing methods are used to identify 5mC regions. AP2 will perform PCR-based amplifications of variable regions (V3, V4) of microbial 16S rRNA coupled with multiplex high-throughput sequencing (NGS) to determine the microbiome. The bioinformatic analysis of this data is carried out continuously in WP3 and in close cooperation with TP7. The data will later be validated in an independent sample collection at the level of the multimarker panel (TP8).
Sub-project 5
The goal of TP5 is the development of biomarkers for patients with curative treatment. In phase 1, patients who cannot be treated curatively are initially included for clinical validation. In phase 1, patients with CRC up to 50 years of age will be screened using dPCR and targeted NGS with plasma samples obtained at the time of diagnosis and before the start of treatment OR at the time of relapse or progression after treatment examined for tumor-specific mutations. In addition, healthy control plasma samples (TP3 & TP9) are analyzed in an analogous manner. Phase 2 will focus on patients with intended curative treatment. Here, plasma samples from CRC patients are analyzed at the following time points: at diagnosis before starting treatment; after completion of treatment and at the time of relapse. In addition, healthy controls (TP3 & TP9) are analyzed.
Sub-project 6
Biological age-based proteome analysis aims to (a) provide biomarkers for (re)establishment of health, (b) uncover early indicators of disease recurrence, (c) define the optimal metabolism of the healthy phenotype and (d) to help distinguish dietary responders from non-responders. The long-term result is personalized nutrition to maintain individual health in young adults with colorectal cancer. TP6 is divided into the following work packages reflecting the different human sample entities:
• WP1: Proteome analysis of serum/plasma samples (TP1 & TP3/TP9)
• WP2: Proteome analysis of organoids (TP3)
• WP4: Proteome analysis of sample preparation experiments (TP8)
For relative protein quantification, LC-ESI-MS/MS is used in data-independent (DIA/SWATH) mode in all APs. DIA/SWATH uses variable mass windows to enable complete measurement of all detectable proteins in a sample, i.e. identification and quantification of thousands of proteins in just one measurement.
Sub-project 7
The goals of TP7 are divided into 4 work packages. WP1 is to set up a central data repository for all research data according to FAIR principles with controlled access, WP2 is to install standardized data analysis. WP3 aims to develop a liquid biopsy-based multi-omics biomarker for the early diagnosis and monitoring of CRC. WP4 aims to support the development of marker panels, nutritional interventions and patient monitoring. In the first phase, years 1-4, TP7 will build the computational infrastructure and focus on the computational development of a CRC biomarker panel based on CRC, control and organoid samples together with clinical data. In the second phase, TP7 will support the translation of the marker panel into clinical practice and provide computational support for nutritional intervention (TP9) and long-term patient monitoring.
Sub-project 9
Screening and optimization for optimal nutrition to prevent biological intestinal aging. In a first step, the influence of various nutritional interventions on the biological age of the intestine will be investigated in an exploratory pilot study. The underlying hypothesis is that biological age is causally involved in the development of CRC. For this purpose, together with TP3, healthy volunteers <50 years will be recruited and included in study 1 (screening). Biomaterials including primary intestinal stem cells for organoid cultivation (TP3) as well as for metabolic characterization (TP3) will be obtained from these subjects. The subjects go through a 14-day, digitally accompanied test phase with test meals and constant glucose monitor (CGM) analysis. This is part of the MillionFriends program offered by the participating KMU Perfood GmbH. This is followed by a 6-month intervention phase (healthy diet according to national guidelines vs. low-GI vs. reduced-calorie intermittent fasting), followed by a final endoscopy including biomaterial collection. The aim is to find out whether and if so, which of the tested nutritional interventions have a positive influence on the biological age in the intestine. The most effective intervention with regard to the primary endpoint serves as a comparison group and as a basis for establishing an optimized diet that takes up the results of TP3.
Study Design: Allocation: ; Masking: ; Control: ; Assignment: ; Study design purpose: prevention
