Danique Heuvelings

335 Summary, general discussion and future perspectives The systematic review described in chapter 11 aimed to evaluate (the quality of) sequencing studies reporting on specific biomarkers in primary colorectal tumors that could serve as a prediction tool to estimate the risk of peritoneal spread. The review identified 17 retrospective cohort studies that reported on potential biomarkers. The DNA analyses showed that most included articles (n = 10) described BRAF mutant tumors to be more likely to have PM and/ or mutations in BRAF were more common in patients with PM compared to those without. Some additional genes were mentioned as possible mutated genes associated with PM by several authors but were, except for one, all investigated in only one study. Broader panel analysis did not show additional discoveries, and RNA outcomes were not consistent either. As almost all included studies were retrospective with a different number of patients, different patients’ characteristics and different used sequencing methods, comparisons between the studies were limited due to this heterogeneity. Unfortunately, most of the studies did not clearly specify whether the authors were using tumors from synchronous or metachronous PM patients. It was therefore hard to distinguish and separate these two scenarios in the results. Based on the given evidence, we concluded that the summarized genes that were possibly associated with PM (especially BRAF), were not reliable enough to function as an individual biomarker in a clinical setting and future biomarkers research in a homogenous population was necessary. Subsequently, our research team performed such an analysis in the next chapter. Subsequently, chapter 12 explored and compared genetic alterations in primary colorectal tumours of patients without metastases, with metachronous PM and with metachronous liver metastases (LM) to potentially discover biomarkers for metachronous PM. This retrospective analysis involved forty patients with T3 stage CRC, categorized into the three metastatic groups based on a 5-year follow-up (M0 = 20, PM = 10; LM = 10). To ensure a homogenous population, patients with any synchronous metastases were excluded. A comprehensive genome sequencing (Trusight Oncology (TSO) 500 analysis) was conducted on primary formalin-fixed paraffin-embedded tumor samples, targeting DNA alterations in 523 genes and RNA fusion transcripts in 55 genes. Two patients were excluded (LM = 1 and PM = 1) from the final analysis, resulting in a final sample size of 38. Microsatellite instability (MSI) was identified in four M0 tumors and one PM tumor. There were new genes identified that had not been described in relation to metachronous PMs, or metastases in general, although the clinical significance remained unknown due to the small sample size. Notably, BRAF p.V600E mutations were exclusively present in PM patients with microsatellite stable (MSS) tumors (37.5%, p = 0.010). The outcome of chapter 12 strengthened the suggestion made in chapter 11 and underscored significance of closely monitoring BRAF p.V600E mutated MSS tumors concerning the emergence of metachronous PM. In current clinical practice, the classification of the MSI status is the only genetic test that is routinely performed in CRC patients to decide adjuvant therapy decisions 80. Other genetic tests, such as BRAF mutation status, are only 14

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