10582 Background: The integration of germline genetic testing, frequently from blood, into routine oncological care has increased the frequency at which pathogenic TP53 mutations are found in a variant allele frequency (VAF) range suggestive of either mosaic Li-Fraumeni Syndrome or clonal hematopoiesis of indeterminate potential (CHIP). Mosaic LFS is a rare and poorly described phenomenon wherein a pathogenic TP53 mutation is dispersed in low levels through multiple somatic tissues after stochastically developing during early embryogenesis. In comparison, CHIP is restricted to the bone marrow niche. Germline mosaicism in Li-Fraumeni Syndrome (LFS) is inferred when genomic DNA from a second source recapitulates the exact same TP53 mutation. Given the challenges with sample acquisition, mosaicism has not been previously described in depth in the medical literature. Methods: Two pediatric patients with LFS-associated cancers (glioblastoma, osteosarcoma & ALL respectively) underwent clinical genetics evaluation and were found to have pathogenic TP53 mutations. Initial genetic testing was performed in a CLIA/CAP environment through a send out test or internally. A broad array of somatic tissues was collected at autopsy with subsequent DNA isolation and amplicon sequencing in triplicate to determine the mean VAF of the pathogenic TP53 allele. Results: For both patients, the initial germline test suggesting mosaic LFS had a VAF between 20-30%. Confirmatory intent fibroblast culture in Patient 1 was reported as negative by an external clinical lab and Patient 2’s VAF was much lower. The VAFs for the rest of the somatic tissues as well as in their corresponding cancers are reported below (Table) as means and standard error of the triplicate. Despite the negative fibroblast culture, Patient 1 did demonstrate mutant TP53 in other tissues, confirming mosaicism. Conclusions: The proportion of pathogenic alleles is highly heterogeneous amongst different somatic tissues in mosaic LFS patients. Although CHIP exclusion through germline testing of cultured skin fibroblasts is common, our patients’ skin samples were amongst the tissues with the lowest VAFs. Mosaicism should be highly suspected when tumor sequencing demonstrates an identical mutation to the putative germline mosaic one even with equivocal fibroblast culture results. [Table: see text]