Abstract Introduction/Objective PD-L1 expression on colon cancer cells is associated with a worse prognosis[CGTLUMB(1] and may be an important selection marker in colon cancer patients for developing or combination immunotherapies in the future. A patient is eligible for targeted therapy if they demonstrate PD-L1 expression on a representative section, with different positivity threshold percentages used in different indications. However, little is known about the spatial heterogeneity of PD-L1 expression within tumors or how sampling bias may impact patient care. Here we report the results of a single institution retrospective study of PD-L1 heterogeneity in colorectal cancer to test the hypothesis that PD-L1 staining is homogenous throughout the tumor microenvironment. Methods/Case Report Long-term tissue storage at a single tertiary care center was queried for all cases of resectable (stage I-IV) colonic adenocarcinoma between 1 Jan 2014 and 1 Oct 2020. Cases with at least 4 formalin- fixed paraffin embedded blocks with tumor were selected; exclusion criteria included neoadjuvant therapy and absence of residual tumor on resection. 8 patients were identified meeting inclusion criteria (mean age 68 17 years, 5:3 M:F). PD-L1 (5H1 monoclonal antibody) immunohistochemistry was performed on corresponding tissue. Percent tumor cells expressing PD-L1 in a representative 1mm2 area at invasive margin and central tumor was quantified for each section on digital slide images acquired on an Aperio CS2 Digital Slide Scanner (Leica Biosystems, Wetzlar, Germany) using QuPath (Version: 0.4.3). Results (if a Case Study enter NA) The mean ± SD of PD-L1 staining was 2.7±4.4% at invasive margin and 0.2±0.25% within central tumor; no statistically significant difference between regions was identified (paired t-test, p=0.15). Notably, 2 of the 8 cases demonstrated ranges at the invasive margin (0-36% and 1.3-35%) that span the cutoff for several commercially available anti-PD-1/PD-L1 agents. Conclusion These findings demonstrate that PD-L1 quantification can vary significantly within the same tumor depending on where counts are performed. This has significant clinical implications as PD-L1 positivity and patient eligibility for targeted therapy is most often assessed in a single location. Our current clinical methods of assessing PD-L1 expression do not account for the spatial complexities of the tumor microenvironment and could contribute to inaccuracy of small volume biopsies.