286 Chapter 12 because of a low rectal primary origin. Other previously described clinicopathological risk factors for the development of metachronous PMs are advanced tumor stage, infiltrative or ulceroinfiltrative tumors, a history of perforation, and obstruction 1, 4, 27. A clinical trial investigating the potential of adjuvant HIPEC in high-risk PM patients, based on these clinicopathological risk factors, showed that adjuvant HIPEC did not improve survival as compared to patients receiving systemic adjuvant chemotherapy 28. In contrast, Arjona-Sánchez et al. concluded that adjuvant HIPEC therapy might be useful in patients with T4 tumors 29. These outcomes suggest that specific biomarkers identified in the primary tumor might be helpful to further estimate the risk of metastatic spread and the need for preventive adjuvant treatments. As our study population has a semi-advanced tumor stage (T3) without (ulcero-)infiltrative or obstructing tumors, we exclude any influence of these possible clinical–pathological risk factors in our current study. DNA and RNA Sequencing The most frequently mutated cancer genes found in our study include APC, TP53, KRAS, SMAD, NRAS, BRAF, PIK3CA, and SOX9. These genes are well known to be involved in the tumorigenesis of CRC 17. Prevalence data in the literature on these well-known oncogenes are in line with our findings 15, 30-51. In addition, 12.5% of our tumors contained MSI, in four cases associated with MLH1 promoter hypermethylation, and in one case with an inactivating MSH6 mutation. This finding is in accordance with the literature, as was the finding that these tumors are often right-sided 52,53, 54. MSI results from the inactivation of the mismatch repair genes (MMR), which leads to the accumulation of somatic mutations, genomic instability, and cancer-associated alterations 37. TMB represents the total number of mutations per Mb found in the DNA of tumor cells and is therefore often significantly higher in MSI tumors. In this study, the five tumors with MSI all had a high TMB (IQR 49.45–Q3 180.60). It has been suggested that MSI status may be useful as a predictor of the risk of developing metachronous CRC, because it can cause a further increase in metastatic potential 30, 52. However, we did not observe a higher incidence of MSI tumors in our CRC cohort that developed metastases. Interestingly, BRAF p.V600E mutations were found to be exclusively present in PM patients with RAS wildtype MSS tumors (37.5%, p value = 0.010). Approximately 10–14% of all CRC cases have BRAF-activating mutations 30, 37, 55. BRAF encodes a serine/ threonine protein kinase, which plays an important role in the mitogen-activated protein kinase (MAPK) pathway. This pathway drives cell proliferation, differentiation, migration, survival, and angiogenesis, and therefore, changes in this pathway are associated with tumorigenesis 55. The BRAF p.V600 mutation, caused by a transversion in exon 15 resulting in a valine amino acid substitution 56, accounts for more than 90–95% of BRAF mutations 37, 55 and is associated with poor overall survival 30. In addition, we identified a FAM198A-RAF1 fusion in one PM sample. Both RAF1 and BRAF belong to the RAF family of protein kinases playing a role in MAPK signaling. Previous studies suggested that BRAF p.V600 mutant tumors are more likely to develop PM 15, 38, 41-43, 57. Therefore, we and some authors recommend analyzing BRAF mutation for its prognostic value in primary T3 CRC 37, 40.
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