CHAPTER 4 82 95ºC for two minutes. Twenty μL was then used for PCR in a total volume of 50μL including a common forward primer, bar-coded reverse primers, and iProof high fidelity master mix (1725310, Bio-Rad, Veenendaal, The Netherlands). The resulting PCR products were pooled and purified with 0.8x volume of Agencourt Ampure XP Beads (A63881, Beckman Coulter, Woerden, The Netherlands). Sequencing and analysis The purified libraries were sequenced on a NexSeq500 instrument (Illumina, San Diego, CA). The Sequence Pilot software (version 4.4.0; JSI Medical Systems) was used to demultiplex the bar-coded reads and create consensus (‘unique’) reads to minimize sequencing errors. Variant calling was performed and variants were annotated as benign, likely benign, unknown, likely pathogenic or pathogenic using publicly available databases such as The Clinical Knowledgebase (CKB, https://www.jax.org/clinical-genomics/ckb), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), Cancer Genome Interpreter (CGI, https:// www.cancergenomeinterpreter.org/home) and the Catalog of Somatic Mutations in Cancer (COSMIC, cancer.sanger.ac.uk/cosmic) 24. The last three categories were considered relevant and consisted of known activating hotspot mutations for the oncogenes25, and frameshift, nonsense, missense, and splice-site mutations for the tumor suppressor genes. The following molecular subgroups were identified based on these sequencing results: POLE mutated, MSI, TP53 wildtype and TP53 mutated. In case of double classifiers (for example, POLE and TP53 mutation), the tumor was classified as previously described. It was previously shown that there is an excellent correlation between TP53 mutational status and p53 IHC, and we therefore decided not to perform additional IHC in this study 26. Statistical analysis For statistical analyses, Statistical Package for the Social Sciences (SPSS), version 25.0 (IBM, New York, NY, USA) was used. Clinicopathological differences between subgroups were compared using the Fisher’s exact test and χ² for discrete variables and the MannWhitney U test for continuous variables. Survival analyses were performed using the Kaplan Meier (KM) curves and univariable and multivariable Cox-regression analysis. A recurrence was defined as first sign of relapse after a 6-month disease-free interval after initial surgery. Disease-free survival (DFS) was calculated from the date of initial surgery until the date of recurrence, whereas overall survival (OS) was calculated from the date of initial surgery until the date of death or, for surviving patients, to the date of last follow-up. Disease-specific survival (DSS) was calculated from the date of primary treatment to the date of death caused by the disease or, for surviving patients, to the date of the last follow-up.
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