3148 Background: Recent proteogenomic analyses of CRC revealed that driver gene alterations are enriched in the endocytosis pathway (Vasaikar S, et al. Cell 2019;177:1035-49). Endocytosis is a cellular system involving post-translational modification of plasma membrane proteins through internalization, intracellular trafficking, degradation, and recycling. Clathrin-mediated endocytosis (CME) is the main endocytic portal, and endosomal sorting complexes required for transport (ESCRT) play a critical role in the lysosomal degradation pathway. Besides the well-known function of endocytosis attenuating signaling pathways through receptor clearance from the cell surface, the opposite function contributing to signal maintenance has also been reported. However, the clinical implications of the endocytosis pathway alterations in CRC are largely unclear. Methods: We retrospectively reviewed CRC patient samples (n = 15025) submitted to a commercial CLIA-certified laboratory (Caris Life Sciences, Phoenix AZ). Next-generation sequencing of DNA and RNA (whole-transcriptome sequencing) and immunohistochemistry (IHC) were performed. CME-related (47 genes) and ESCRT-related (35 genes) expression signatures were calculated as composite z-scores and compared between subgroups stratified by RAS/ BRAF mutation status, MSS/MSI status, tumor sidedness, and consensus molecular subtype (CMS). VPS4A/ VPS4B expression correlation with major oncogenic pathway signatures (composite z-scores) and CMTM6/ CMTM4/ HIP1R expression association with PD-L1+ IHC were also assessed. Results: Among 17 endocytosis-related genes, no pathogenic/likely pathogenic mutations were identified. The CME-related signature was increased in RAS/ BRAF wild type vs. mutant (0.93 z-score difference, p= 0.04) and MSS vs. MSI-high (6.0 z-score difference, p< 0.01), while the ESCRT-related signature was higher in MSS compared to MSI-high (2.7 z-score difference; p< 0.01). No differences between tumor sidedness were observed in both CME- and ESCRT-related signatures (0.81 and 1.17 z-score differences, respectively). CMS4 had the highest expression of both signatures, while CMS3 had the lowest, of both CME- and ESCRT-related genes (each > 20 z-score difference, p< 0.01). VPS4A and VPS4B expression had a strong positive correlation with WNT, EGFR/MAPK, TGF-beta, and Notch pathway signatures (0.65-0.83 Spearman, all p< 0.01). CMTM6 expression was positively associated with PD-L1+ IHC (1.2-fold increase vs PD-L1-negative, p< 0.01), while CMTM4 and HIP1R expression showed a negative association (0.7- and 0.9-fold decrease, respectively, p< 0.01). Conclusions: This large study indicates endocytosis pathway expression is positively associated with oncogenic pathway signaling in CRC. Further analysis of RAS/ BRAF wild type, MSS, and CMS4 patient subgroups are warranted to determine the efficacy of targeting endocytosis pathways in CRC.