279 Predictive genetic biomarkers for the development of peritoneal metastases in colorectal cancer Table 1. In- and exclusion criteria for patient selection. Inclusion Criteria Exclusion Criteria • Tumor histological type defined as an adenocarcinoma • Pathology report confirmed a radical resection with >15 lymph nodes • Pathological T3 classified according to the TNM classification • M0 group: Follow-up of 5 years without development of metastases • Acute colorectal surgery with blowout or proven perforation • Anastomotic leak after surgery • Patients with hereditary CRC • LM and PM group: Metachronous metastases > 6 months after primary surgery M0: no metastases; LM, liver metastases; PM, peritoneal metastases. Tumor Samples Primary tumor FFPE tissue samples were obtained from MUMC+ and CZE. From each FFPE tissue specimen, 10 paraffin sections of 5 μm were cut. Hematoxylin and eosin (H&E) staining was performed. An experienced pathologist (J.B.) marked the tumor circumflex and estimated the tumor cell percentage under the microscope. Only samples with ≥10% tumor cell percentage were considered eligible for further analysis. Subsequently, microdissection with a pointed surgical blade was performed. DNA and RNA were extracted and isolated using a Maxwell RSC® System for Genomic DNA or RNA Extraction with a FFPE AS1450 kit and FFPE AS1440 kit, respectively (Promega, Madison, WI, USA). A blank control sample was analyzed in parallel to each set of samples. A minimum amount of 40 ng DNA or RNA was necessary for further analysis. DNA samples were stored at 4 °C and RNA samples at −80 °C. Fragment analysis of both DNA and RNA samples was performed as quality control. For DNA, a PCR was performed to visualize all DNA fragments. For RNA, the samples were assessed using a 4150 TapeStation system, which separates nucleic acids through electrophoresis. All fragments needed to be at least 200 bp in length. TruSight Oncology 500 Analysis TSO500 is an NGS assay that enables the comprehensive genomic profiling of tumor samples. The TSO500 panel (20028216; Illumina, Hayward, San Diego, CA, USA) was used to detect mutations and identify other relative pan-cancer genes in the tumor samples, as previously described by Verkouteren et al. 21. The analysis includes 523 genes for mutations (all for single-nucleotide variants (SNVs)) and 59 for copy number variations (CNVs) (amplifications, insertions, and deletions). In addition, the assay allows for the identification of MSI and TMB. Besides DNA analysis, 55 genes are screened for fusion and splice variants on the RNA level. All genes included in the TSO 500 panel can be found in Supplementary Section S1, Figure S3. DNA and RNA processing and the generation of library preparations were performed according to the manufacturers’ instructions. Data analysis was performed using the TSO500 Local App (Illumina, Hayward, San Diego, CA, USA). For DNA analysis, additional thresholds were maintained. First, for variant allele frequency, a percentage of ≥5% was maintained. Second, for classification as an amplification, a fold change of ≥3 was maintained. Third, the threshold for classification as MSI-high was ≥20% of microsatellite sites being unstable. 12
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