Microsatellite instability (MSI) in TCGA qualified pancreatic adenocarcinomas with 20% or greater tumor nuclei was evaluated by the Biospecimen Core Resource at Nationwide Children’s Hospital (TCGA Center 23). Percent tumor nuclei was assessed through pathologists review of a frozen histological section of the tumor immediately adjacent to the tissue that gave rise to the DNA analyte. The MSI-Mono-Dinucleotide Assay was performed to test a panel of four mononucleotide repeat loci (polyadenine tracts BAT25, BAT26, BAT40, & transforming growth factor receptor type II) & three dinucleotide repeat loci (CA repeats in D2S123, D5S346, & D17S250). Two additional pentanucleotide loci (Penta D & Penta E) were included in this assay to evaluate sample identity. Multiplex fluorescent-labeled PCR & capillary electrophoresis was used to identify MSI if a variation in the number of microsatellite repeats was detected between tumor and matched non-neoplastic tissue or mononuclear blood cells. Equivocal or failed markers were re-evaluated by singleplex PCR. Tumor DNA was classified as microsatellite-stable (MSS) if zero markers were altered, low level MSI (MSI-L) if less than 40% of markers were altered and high level MSI (MSI-H) if greater than 40% of markers were altered. In the MSI-Mono-Dinucleotide Assay, this equated to MSI-L classification if one or two markers were altered, and MSI-H if three to seven markers were altered. Penta D and E markers were scored in the same manner as the MSI markers indicated below, however they did not contribute to MSI Class calculation. Any qualified pancreatic adenocarcinoma with less than 20% tumor nuclei was classified as indeterminate due to this being the limit of sensitivity for the MSI-Mono-Dinucleotide assay. However, caution should be used when interpreting MSI results from any pancreatic adenocarcinoma found to be MSS by this assay. The gross distribution of tumor nuclei in pancreatic adenocarcinoma has a high degree of spatial heterogeneity, therefore the actual percent tumor nuclei within the extracted tissue may be lower than that found in the pathologist reviewed adjacent section. As a result, the MSS calls from this assay may reflect a low degree of tumor content within the extracted sample, rather than the absence of MSI within the potentially small amount of tumor contained within.