266 Chapter 11 Techniques In the studies evaluated in this review, several different genetic research techniques were applied. Since most studies used targeted PCR techniques to detect specific gene mutations, the number of studies that used comprehensive genetic analyses was scarce. The development and use of NGS technologies have revolutionized the speed and throughput of DNA and RNA sequencing 64, 65. However, since the number of relevant cancer genes guiding targeted therapy in CRC is still limited and costs per sample are substantial, NGS sequencing is not yet commonly used in clinical decision making or limited to mutation hotspot target regions 66. This has most likely influenced the research to unmap PM predictive biomarkers so far, and we believe that more comprehensive NGS analyses are needed for this purpose. When we critically look at the choice of techniques used in the included studies, we believe these were too restricted to identify DNA/RNA biomarkers in the primary tumor of CRC patients with synchronous or metachronous PM. As mCRC is a highly complex genetic disease, an understanding of how all aspects interact is required to achieve the prediction and treatment of colorectal PM. Single target techniques, mostly used in the included articles in this paper, might be insufficient for this purpose. We believe that omics techniques (i.e., techniques that generate high-throughput data 67) might be a promising method for new CRC biomarkers research instead of most of the methods used in this paper. The integration of multiple omics techniques, by combining genomic data with data from other modalities such as transcriptomics, epigenetics, and proteomics, to measure gene expression, gene activation, and protein levels, could be helpful to reveal this problem in further research. This integration might bring us much closer to the prediction, prevention and tailored treatment of PM in CRC 68. Limitations This is the first systematic literature review of DNA/RNA biomarkers in relation to colorectal PM to the author’s knowledge. This study has also some limitations. First, almost all included studies were retrospective with a different number of patients and different patients’ characteristics (T-stage, number of metastatic sites, treatments, etc.). Second, comparisons between the studies are limited due to heterogeneity, and a meta-analysis was therefore not possible to perform. The standardization of techniques and analysis and more insight in the individual analysis outcomes via FAIR data sharing would be helpful. Third, most studies focused on the most commonly analyzed CRC target genes, i.e., KRAS, NRAS, BRAF, PIK3CA, and TP53 with simple sequencing methods and PCR technology. Only three studies performed a broader gene panel NGS analysis. Fourth, most of the included studies did not report if CRC patients received neoadjuvant systemic treatments and if they did, which type. Such treatments could namely affect the outcomes of the genetic analysis. Fifth, most of the studies lacked the MSI of the CRCs. Sixth, all studies showed a moderate to high risk of bias with a high risk for the confounding domain.
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