Colic Flexure, Right

Samples were collected from an IBD cohort at St. Olav’s University Hospital, Trondheim, Norway. Study participants were patients admitted to the Department of Gastroenterology for colonoscopy. Participants were either diagnosed with UC or CD, or were admitted for diagnostic colonoscopy due to symptoms unrelated to IBD. Patients were only included as normal controls after all clinically indicated examinations had concluded no signs of gastrointestinal disease. In the IBD groups (UC and CD), four endoscopic pinch biopsies were taken from macroscopically maximally inflamed mucosa, as well from the hepatic flexure in cases where this was found to be macroscopically un-inflamed UC unaffected (UCU) and CD unaffected (CDU)). Two UCU samples, 228 F and 118 F were obtained from ascending colon and rectum, respectively. For the normal (N) group, four biopsies were taken from the hepatic flexure. One biopsy from each area was fixed in 4% buffered formaldehyde, while the three remaining biopsies were snap frozen and stored in liquid nitrogen.

The Cancer Genome Atlas (TCGA) data (TCGA-COAD and TCGA-READ datasets) and NCBI GEO (GSE190826 dataset) were used to examine the expression of SPP1, S100A4 and SPARC and to perform survival analysis in colorectal cancer patients. TCGA data included information about SPP1, S100A4 and SPARC expression, that was evaluated in the following groups of patients: a) with colorectal cancer (common group) (N=417), b) with colon cancer, including transverse colon, ascending colon, descending colon, sigmoid colon, cecum, hepatic flexure, splenic flexure (n=305), c) with rectal cancer, including rectosigmoid junction and rectum (N=112), with available clinical information and records on recurrence and survival rates (in details in Supplementary Table S1). Patients with advanced stage IV were excluded. GSE190826 dataset included 92 patients with rectal cancer treated with neoadjuvant chemoradiotherapy (NCRT); information about pre-treatment levels of SPP1, S100A4 and SPARC mRNA expression was obtained. The TCGA biolinks was used for retrieving RNA-seq data from the GDC database. The raw sequencing reads were processed via the DESeq2 R package. The raw counts were depth normalized and variance stabilized via the variance stabilizing transformation (VST) for downstream survival analysis.

The EPIC cohort includes over 520,000 individuals who were recruited between 1992 and 2000 from 23 study centers across 10 European countries (Denmark, France, Germany, Greece, Italy, Norway, Spain, Sweden, the Netherlands, and the UK). Participants were 35-70 years of age at recruitment, and approximately 70% of the cohort are women. The study design has been previously described [22 (link), 23 (link)]. In brief, extensive questionnaire data on dietary and lifestyle variables were collected at baseline, and approximately 75% of individuals provided non-fasting blood samples.
Incident cases of colorectal cancer were identified through record linkage with regional cancer registries or via a combination of methods, such as the use of health insurance records, contacts with cancer and pathology registries, and active follow-up through participants and their next of kin. Colorectal cancer was defined using the tenth edition of the International Classification of Disease (ICD-10) and the second edition of the International Classification of Disease for Oncology (ICD-O-2). Proximal colon cancers included those found within the cecum, ascending colon, hepatic flexure, transverse colon, and splenic flexure (C18.0 and C18.2–18.5). Distal colon cancers included those found within the descending (C18.6) and sigmoid (C18.7) colon. Overlapping (C18.8) and unspecified (C18.9) lesions of the colon were classed as colon cancers only. Cancer of the rectum included cancers occurring at the recto-sigmoid junction (C19) and rectum (C20).
The current study employed a fasted subset of EPIC data, obtained from two separate metabolomics studies on colorectal cancer, as a discovery cohort. Samples were analyzed using the Biocrates AbsoluteIDQ^TM p180 kit (467 cases and 467 matched controls) and the p150 kit (1141 cases and 1141 controls). Combining these studies and then excluding non-fasting participants resulted in a final combined sample of 654 fasted cases and 654 controls, of which 354 case-control pairs were analyzed using the p180 kit. Controls were selected using incidence density sampling from all cohort members who were alive and free of cancer (except non-melanoma skin cancer) at the time of diagnosis of the colorectal cancer cases. Controls were matched to cases on age at recruitment (within 6 months), sex, study center, follow-up time since blood collection, time of day at blood collection (within 4 h), and fasting status. Women were further matched on menopausal status (pre-, peri-, and post-menopausal) and, in pre-menopausal women, phase of menstrual cycle at blood collection. Approval for the study was obtained from the International Agency for Research on Cancer (IARC) and local center review boards. All participants provided written informed consent.