Increased adoption of personalized medicine has brought comprehensive genomic profiling (CGP) to the forefront. However, differences in assay, bioinformatics, and reporting systems and lack of understanding of their complex interplay are a challenge for implementation and achieving uniformity in CGP testing. Two commercially available, tissue-based, in-house CGP assays were compared, in combination with a tertiary analysis solution in a research use only (RUO) context: the AVENIO Tumor Tissue CGP RUO Kit paired with navify Mutation Profiler (RUO) software and the TruSight Oncology 500 RUO assay paired with PierianDx Clinical Genomics Workspace software. Agreements and differences between the assays were assessed for short variants, copy number alterations, rearrangements, tumor mutational burden, and microsatellite instability, including variant categorization and clinical trial-matching (CTM) recommendations. Results showed good overall agreement for short variant, known gene fusion, and microsatellite instability detection. Important differences were obtained in tumor mutational burden scoring, copy number alteration detection, and CTM. Differences in variant and biomarker detection could be explained by bioinformatic approaches to variant calling, filtering, tiering, and normalization; differences in CTM, by underlying reported variants and conceptual differences in system parameters. Thus, distinctions between different approaches may lead to inconsistent results. Complexities in calling, filtering, and interpreting variants illustrate key considerations for implementation of any high-quality CGP in the laboratory and bringing uniformity to genomic insight results., Competing Interests: Disclosure Statement H.-P.A., F.F., and U.S. are employees of Signature Diagnostics GmbH (H.-P.A. at the time of the analysis, but now retired). M.C.H., J.O., and J.M.A. are employees of Foundation Medicine, Inc. (J.O. at the time of the analysis, but is no longer an employee), and M.C., S.O., and T.W. are employees of Foundation Medicine GmbH; all hold stock in F. Hoffmann-La Roche Ltd. K.H. is an employee of Helios MVZ Pathologie Erfurt GmbH. M.T. is an employee of F. Hoffmann-La Roche AG and holds stock in F. Hoffmann-La Roche Ltd. D.G. holds stock in Univ8 Genomics Ltd.; has received honoraria (institution) from Roche, Illumina, Novartis, AstraZeneca, Eli Lilly, Amgen, and Janssen; has participated in a consulting or advisory role (institution) for AstraZeneca and Novartis; has participated in a speakers' bureau (institution) for AstraZeneca; and has received travel, accommodations, and expenses (institution) from Roche, AstraZeneca, and Illumina. E.P. received compensation for speaking engagements and advisory roles, with payments made directly to his affiliated university, from Roche, AstraZeneca, Novartis, and MSD. H.S. and M.D.V. are employees of Roche Diagnostics Solutions, Inc., and M.D.V. holds stock in F. Hoffmann-La Roche Ltd. E.S. reports lectures for Bio-Rad, Seracare, Novartis, Roche, Biocartis, Illumina, Eli Lilly, Janssen-Cilag, Pfizer, AstraZeneca, and Agena Bioscience; he is consultant in advisory boards for MSD/Merck, GSK, AstraZeneca, Astellas Pharma, Sysmex, Roche, Pfizer, Novartis, Bayer, Bristol Myers Squibb, Eli Lilly, Amgen, Biocartis, Illumina, Agena Bioscience, Janssen-Cilag (Johnson & Johnson), Sinnovisionlab, Diaceutics, and CC Diagnostics; and received research grants from Pfizer, Biocartis, Invitae-ArcherDX, AstraZeneca, Agena Bio-science, Bristol Myers Squibb, Bio-Rad, Roche, Boehringer Ingelheim, CC Diagnostics, and Abbott (all paid to University Medical Center Groningen account); and travel reimbursements from Bio-Rad, Abbott, Illumina, Agena Bioscience, and Roche. All authors received support in the form of third-party writing assistance for this manuscript, furnished by Nucleus Global, an Inizio company, and provided by F. Hoffmann-La Roche Ltd./Roche Diagnostics Solutions, Inc., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)