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The development of personalized medicine in digestive oncology is based on the detection of biomarkers that are predictive of the response to targeted treatments. The ability to detect these biomarkers under different conditions is therefore crucial. This review will focus on technical aspects in this context: the quality of the sample to be analyzed (tumor tissue or liquid biopsy), methods for detecting complex abnormalities (gene fusions, mutation signatures), and next-generation sequencing (NGS) with different panels of genes. It will also discuss the new biomarkers to be aware of in digestive oncology in 2025 and their clinical indications: claudin 18.2, FGFR2 alterations, HER2 status, polymerases POLE and POLD1 mutations, and targeting KRAS mutations. The role of ctDNA in digestive cancers and that of large-scale NGS sequencing, including whole genome sequencing, will also be discussed.