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Introduction In colorectal cancer (CRC), the use of immunotherapy is currently based on the evaluation of microsatellite instability (MSI) for the identification of patients who may benefit from this treatment. Recently, tumour mutation burden (TMB) has been evaluated as a useful biomarker to predict response to immune checkpoint (IO) therapy. TMB can be measured by means of targeted next generation sequencing with panels covering approximately 1 Mb of the human genome. We performed TMB analysis on tumor cell lines and CRC samples and compared the results with MSI status to explore the relationship between these two biomarkers. Materials and methods MSI status was evaluated by means of routine immunohistochemistry (IHC), the Idylla MSI assay (Biocartis) and with fluorescence capillary electrophoresis-based DNA fragment size analysis of a T17 mononucleotide sequences within intron 8 of heat shock protein 110 kDa HSP110 (17 polythymine, T17). TMB evaluation was performed with the Oncomine Tumor Mutation Load Assay (Thermofisher) on the Ion S5XL platform. Data analysis was carried out using Ion Reporter Software v5.6. TMB was calculated as the total number of somatic single nucleotide variants (SNVs) divided by number of bases with sufficient coverage. Results We first evaluated the TMB in 13 tumor cell lines and correlated the results with massively parallel sequencing data from >1600 genes for the same cell lines available on cBioPortal, demonstrating an high correlation between the Oncomine Tumor Mutation Load Assay and the TMB value obtained by wide genetic profiling (R² = 0.976). We next analyzed a cohort of 23 MMR deficient (MMR-D) and 32 MMR-proficient (MMR-P) formalin fixed paraffin embedded (FFPE) CRC, as classified by IHC. The MMR data were compared to TMB and revealed TMB values not in line with the expected results, since some samples in the MMR-D group had low TMB values (TMB range: 5.5-134.4). We re-evaluated the 23 MMR-D samples by means of the Idylla System. The Idylla MSI assay re-classified 7 samples as microsatellite stable (MSS) and categorized the remaining 16 samples as MSI-High (MSI-H). The results were confirmed by the T17 analysis. In the MSS group (n. 39), the mean and median TMB value were 11.48 and 10.39, with TMB ranging from 4.88 to 33.29. The mean and median TMB for the MSI-H group (n. 16) were 43.89 and 38.63, and the TMB values ranged from 19.63 to 134.4. While the medians of the two groups were significantly different (Mann-Whitney test P Conclusions These data suggest that routine IHC methods for MSI evaluation can lead to a wrong classification of CRC. On the other hand, TMB assessment might better identify patients who may benefit from IOs within both the MSI-H and the MSS subgroups. Citation Format: Francesca Fenizia, Raffaella Pasquale, Cristin Roma, Francesca Bergantino, Anna Maria Rachiglio, Nicoletta Chicchinelli, Paolo Graziano, Gerardo Botti, Fabiana Tatangelo, Giosuè Scognamiglio, Christopher Allen, Matilde Lambiase, Alessia Iannaccone, Nicola Normanno. Tumor mutation burden and microsatellite instability in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3975.