280 Chapter 12 Fourth, a threshold of ≥15 mutations per megabase (mut/Mb) was used to define high TMB. Variants were classified subsequently using the inline Varsome application (access via https:// varsome.com). Only pathogenic and likely pathogenic variants were included for further analysis; variants of uncertain significance (VUSs) were excluded. Statistical Analysis Gene mutation frequencies and associations between the found mutations and pathological patient characteristics were estimated. Analysis of both the total cohort (with MSI samples) as well as MSS samples only were performed. Numerical variables were presented as medians with interquartile range (IQR) as appropriate. For categorical variables, the number of patients and percentage were used. To evaluate the statistical significance of numerical variable differences observed between groups, non-parametric tests (Kruskal–Wallis and Mann–Whitney U-tests) were applied. Differences in categorical variables were tested using the Fisher–Freeman–Halton test and Fisher’s exact test. Bonferroni correction for multiple comparison was applied to significant outcomes. All tests were two-sided, and differences were considered significant when the p value was <0.05. All the statistical analyses were performed with SPSS (IBM SPSS Statistics for Apple, Version 27, Armonk, NY, USA). In addition, an analysis with protein analysis through evolutionary relationships (PANTHER) was performed 22. The latter was performed for Gene Ontology molecular functions and biological processes 23, 24, for PANTHER pathways, and for Reactome pathways 25. For each sample, the significantly enriched terms were extracted for subsequent analyses using R (R core team, version 4.2.0., Vienna, Austria). Analysis and visualization of the genetic outcomes were carried out with Python (Matplotlib v3.7.0, Salt Lake, UT, USA) and GraphPad Prism (GraphPad software for Apple, version 8.0.0, San Diego, CA, USA). RESULTS Study Cohort Initially, 40 cases were selected according to predefined in- and exclusion criteria. After revision of the CT, one of the patients was diagnosed with a synchronous metastatic lesion in the lung and excluded from further analysis. All patient characteristics and clinicopathological variables are summarized in Table 2. Most patients were males (64%), with a median age of 69 years (61.00–74.00) at the time of diagnosis of the primary colorectal tumor. There was an overall significant difference for differentiation grade (p value < 0.001) and neoadjuvant treatment (p value = 0.039). After pairwise comparison, a significant difference was found in the differentiation grade when primary tumors of patients with metachronous PM were compared to patients without metachronous metastases (M0) and with metachronous liver metastases (LM) (p value < 0.001 and 0.015, respectively). Patients in the LM group were more often treated with neoadjuvant therapy compared to the M0 group (p value = 0.030), which did not remain significant after Bonferroni correction.
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