Abstract Introduction: Next Generation Sequencing (NGS) is transitioning from research to routine in clinical practice for hematological malignancies. Data analysis and annotation of variants are significant barriers to NGS adoption. NAVIFY® Mutation Profiler (NMP) is a CE-marked*, cloud-based tertiary analysis software that provides curation, annotation, and reporting of somatic genomic alterations and biomarker signatures identified by NGS. It provides information on genomic variants based on published biomedical literature, public genomic databases, medical guidelines, drug labels, and results of clinical trials. The software leverages classification guidelines based on AMP, to provide information on detected somatic genomic variants, and inform on associated therapies according to region specific approvals. To validate accuracy and reproducibility of the NMP software and curation process for hematologic malignancies, a clinical evaluation study was performed. Methods: NMP 2.1 was used as a tertiary analysis software. A total of 86 variants derived from hematologic malignancies (including myeloid and lymphoid leukemias, B cell lymphomas and multiple myeloma), contrived as 12 VCF files were generated. These comprised of the following classes of genomic alterations: single nucleotide variants, insertions and deletions, fusions, copy number alterations, and indels. Out of 86 variants 42 were Tier 1A and 44 were non-Tier 1A, based on AMP classification. The study was performed at 4 external sites with 7 software users (molecular genetics experts). Users uploaded the VCF files into the NMP software, and processed them with instructions provided. NMP reported AMP tier of variants and associated therapies were evaluated for agreement among the users. Results: For the reproducibility, 100% agreement for the hematologic malignancy cases was achieved for each agreement analysis, demonstrating software users can produce the exact same output, given the same files and condition variables entered into the software. For accuracy, tier classification agreement was 91.34% for Tier IA and 95.02% for all hematologic variants. The agreement on associated therapies for the NMP classified Tier IA hematologic variants was 99.08%. Conclusions: NAVIFY® Mutation Profiler is a robust automated solution for genomic variant reporting of hematologic malignancies. The tier classifications and available therapies resulting from the NMP annotation and curation are highly concordant with molecular genetics experts. With the increasing number of known clinically relevant genomic variants in hematologic malignancies, software capable of automatically identifying and accurately classifying somatic genomic variants, promises to decrease the manual review time necessary in clinical practice. *CE-IVD. United States: Research Use Only. Not for use in diagnostic procedures. Citation Format: Ruby Singhrao, Lisha Capucion, Shikha Chugh, Shuba Krishna, Adama Parham, Amy Harrell, Ranga Yerram, John Duncan, Manana Javey. Clinical evaluation of somatic genomic alteration annotation for hematological malignancies using tertiary analysis software [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5236.