282 Chapter 12 DNA Sequencing In one LM sample, no (likely) pathogenic mutations were found, most probably due to the low residual tumor area after neo-adjuvant treatment. This outcome was considered unreliable, and the sample was excluded from further DNA analysis. The final study cohort thus consisted of 38 patients (Supplementary Section S1, Figure S1 and Table S1). Microsatellite instability (MSI) analysis showed that a total of 5/38 samples (four M0 [20%] and one PM [11%]) were MSI with a median of 53.91% unstable MSI sites (Q1 32.55–68.11; Supplementary Section S1, Figure S2). These samples also showed a significant TMB with a median of 64.3 mut/Mb (Q1 49.45–Q3 180.60). All significant MSI and TMB patients had a right-sided primary tumor with poor or poor/moderate differentiation grade. The occurrence of MSI and TMB was not significantly different between the three groups. One of the MSI samples harbored a nonsense mutation in MSH6 (i.e., c.3772C>T p.(Q1258*)), a DNA mismatch repair protein, which could explain the instability of the sample. All other four samples showed MLH1 promotor hypermethylation. Mutational signatures from each sample were individually analyzed. Base substitution of C>T and T>C were the most common ones in all samples. No specific profile was identified when comparing the three subgroups. A general overview of all variant type frequencies and amplifications is displayed in Supplementary Section S1, Table S2 and of all tumor mutations and amplifications in Table S3. Analysis of the total cohort did not identify significant gene mutations in the PM group nor other subgroups. As MSI samples showed a lot of passenger genes that were influencing analysis outcomes, all MSI samples were excluded for a separate analysis with only microsatellite stable (MSS) tumors. The analysis of the total cohort (MSI + MSS samples, n = 38) can be found in Supplementary Section S2 (Figure S4-5, Table S5). MSS Samples Analysis All MSI tumors were excluded for a separate analysis. This resulted in a study population of 33 patients with MSS tumors (M0 N = 16, LM N = 9, and PM N = 8). A total of 164 (likely) pathogenic genetic alterations were detected in 78 genes (Figure 1). Missense, frameshift, and nonsense mutations were most commonly detected. When comparing the occurrence of all variant types, no significant differences were found. The distribution among cancer genes related to CRC was investigated (Figure 2). APC mutations occurred most frequently; in 4/8 (50%) of the PM cases and 8/9 (89, 89%) LM and 14/16 (87, 50%) M0 patients (not significant). BRAF (c.1799T > A p.(V600E) exon 15) mutations were only present in PM patients in this cohort (3/8 = 37.5%, p value = 0.010). None of the M0 samples were carrying PIK3CA mutations after MSI exclusion, and none of the PM samples were carrying NRAS mutations, although these findings were not significantly different. A detailed overview of all MSS subgroup comparisons with statistical p values can be found in the Supplementary Section S1, Table S4.
RkJQdWJsaXNoZXIy MTk4NDMw