**5. Microsatellite instability and immune checkpoints inhibitors**

Microsatellite instability (MSI) is currently a key biomarker in CRC, with diagnostic, prognostic, and therapeutic implications. For these reasons, MSI analysis is becoming increasingly important and testing for deficient mismatch repair (d-MMR)/MSI is recommended, both for hereditary syndrome screening and due to prognostic and treatment implications [122].

Inactivation of a DNA mismatch repair (MMR) gene (MLH1, MSH2, MSH6, or PMS2) by mutation or transcriptional silencing results in deficient function of the MMR system, responsible for excising DNA mismatches introduced by DNA polymerase during cell division. This activity loss translates in an accumulation of DNA replication errors and mismatches in repeated sequences, leading to hypermutated tumors [123]. In most cases, d-MMR and MSI arise due to sporadic somatic hypermethylation of MLH1 and other genes, but they can also result from germline mutations in MMR genes and from Lynch syndrome in approximately 3% of all CRCs [124].

The MMR system can be assessed through different approaches, as IHC, polymerase chain reaction (PCR)-based assays, and more recently NGS. IHC looks at MLH1, MSH2, MSH6, and PMS2 staining in tumor samples to identify the protein expression loss that characterizes d-MMR [125]. PCR amplification requires both tumor and matched normal samples. Five microsatellite loci have been PCRamplified and analyzed by capillary electrophoresis. Instability at more than one locus was defined as MSI-high (MSI-H), at a single locus as MSI-low (MSI-L), and absence of instability at any locus as microsatellite stable (MSS), proficient MMR

(p-MMR) [126]. NGS detection directly targets certain genes, which are genome sequenced to retrieve information on MSI and MMR and tumor mutational burden (TMB), integrating all information in the same test. NGS requires a smaller sample and is more accurate than PCR. Ethical issues may arise with the use of this technique regarding counseling and consent for additional genetic testing [127]. In CRC, MSI varies according to tumor stage, with higher incidence reported in early stages (20% in stages I-II, 12% in stage III) and lower incidence reported in the metastatic setting (4–5%) [128].
