Clonal Hematopoiesis and Mosaicism Revealed by a Multi-Tissue Analysis of Constitutional TP53 Status

Background: Though germline TP53 pathogenic/likely path-ogenic variants (PV) are associated with Li-Fraumeni syndrome, many detected by multigene panels represent aberrant clonal expansion (ACE), most due to clonal hematopoiesis (CH). Discerning ACE/CH from germline variants and postzygotic mosaicism (PZM) is critically needed for risk assessment and management.Methods: Participants in the Li-Fraumeni & TP53 Understand-ing & Progress (LiFT UP) study with a TP53 PV were eligible. Demographics, personal/family cancer history, and clinical labo-ratory test reports were obtained. DNA from multiple tissues was analyzed using a custom QIAseq assay (ACE panel) that included TP53 and other CH-associated genes; the ACE panel and eyebrow follicles were assessed in a workflow to discern TP53 PV clinical categories.Results: Among 134 participants there was a significant differ-ence for the age at diagnosis (P < 0.001), component cancers (P = 0.007), and clinical testing criteria (P < 0.001), comparing germline with PZM or ACE. ACE panel analysis of DNA from 55 sets of eyebrow follicles (mean 1.4 ug) and 36 formalin-fixed, paraffin imbedded tissues demonstrated low variance (SE, 3%; P = 0.993) for TP53 variant allele fraction, with no significant difference (P = 0.965) between tissue types, and detected CH gene PVs. Of 55 multi-tissue cases, germline status was confirmed for 20, PZM in seven, ACE for 25, and three were indeterminate. Addi-tional CH variants were detected in six ACE and two germline cases.Conclusions: We demonstrated an effective approach and tools for discerning germline TP53 status.Impact: Discernment of PZM and TP53-driven CH increases diagnostic accuracy and enables risk-appropriate care.