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

Abstract

Background: Though germline TP53 pathogenic/likely pathogenic 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 Understanding & Progress (LiFT UP) study with a TP53 PV were eligible. Demographics, personal/family cancer history, and clinical laboratory 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 difference 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. Additional 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.

Figure 1.

Multi-tissue workup of TP53 pathogenic variants (PV) identified in germline (blood/saliva) commercial testing. This diagram depicts a diagnostic flowchart for establishing the presence/absence and variant allele fraction (VAF) of TP53 PVs in multiple tissues, often representing different embryonic germ layers, to discern and subclassify germline status including parental gonadal mosaicism, “de novo” germline status, post-zygotic mosaicism (PZM), or clonal hematopoiesis (CH) as ACE in a single tissue compartment. CH is further differentiated into clonal hematopoiesis of indeterminant potential (CHIP) if the complete blood count (CBC) is normal or extant hematologic neoplasia if not, and the approach for prospective evaluation of clonal evolution and genomic drivers of CH, and clinical state change is outlined.

Figure 2.

Variant allele fraction (VAF) of a TP53 pathogenic variant tested on different platforms. The custom The QIAseq^® amplicon-based 81-gene panel (ACE Panel) was used for assessment of VAF across blood samples from 14 participants. Test-retest variability (reproducibility) was assessed in triplicate. TP53 VAFs detected on the ACE panel were highly reproducible, as the within-participant standard error was 3%, with no statistically significant variability by ANOVA (p = 0.993). There was consistency/correlation of VAF across samples as well as across platforms (commercial laboratory multigene panel testing (MGPT); custom Agilent Hybrid capture panel) (p=0.895). Sanger sequencing values noted but not included in analysis as a semi-quantitative method. Note that Table 3 lists additional CH driver variants detected by the ACE panel, wherein, the test/retest VAF consistency is also shown for these secondary CH variants.

Figure 3.

Clinical category assignment for individuals with a TP53 pathogenic variants. The figure depicts the overall proportion of the clinical categories (germline, ACE/CHIP, PZM) found in this clinic based genetic testing cohort. Based on cascade/transmission testing, clinical criteria, and/or multi tissue analyses, 89/134 (66%) cases had evidence for germline, 26/134 (19%) were categorized as ACE, and 7/134 (5.2%) had evidence for PZM. Additional tissue or transmission data would be needed to discern PZM from broader ACE category for the 12/134 (9%) that were indeterminate.