Abstract

6546 Background: Cellular therapy is a promising approach for many diseases but is associated with a risk of subsequent myelodysplastic syndrome and/or acute myeloid leukemia, also known as therapy-related myeloid neoplasms (tMN). This phenomenon has recently been described with allogeneic hematopoietic cell transplantation (HCT) and gene therapy for sickle cell disease (SCD). Mutation(s) in functional domains of TP53 are often associated with the development of tMN but currently no prospective screening or monitoring technology is available. Methods: A next-generation sequencing (NGS) assay with unique molecular identifiers (UMI) and bioinformatic noise-suppression was designed to discover somatic mutations at a variant allele frequency (VAF) 1% throughout the TP53gene. The background error profile at each base position was calculated using blood genomic DNA from healthy donors. The minimal detectable allele fraction with 95% confidence (MDAF) was calculated for all reported known AML/tMN TP53 hotspots. Orthogonal validation was performed using droplet digital PCR (ddPCR). Results: The median error corrected sequencing depth at 62 known TP53 hotspot regions was 14,512 (95% CI: 14,769 – 15,430) with a theoretical median de novo NGS variant discovery limit of 0.058% (range: 0.037% - 0.13%). Experimental evidence of assay performance characteristics was produced using serial dilutions of 4 missense, 2 splicing, and 1 frameshift TP53 variants with 100% discovery specificity observed (lowest detected VAF range: 0.05 – 0.12%). High correlation was observed in quantification using NGS discovery and ddPCR validation ( r = 0.958, p<0.0001). Pre- and post-HCT clinical samples from 19 patients with SCD, 11 of which had graft failure including 4 who developed tMN, were screened for TP53 mutations. In all tMN cases, the AML-associated TP53 variant was detectable at least 6 months and up to 2.5 years prior to clinical diagnosis of myeloid malignancy. In contrast, no pathogenic TP53 variants were detected in 15 SCD patients who underwent HCT but did not develop tMN. Conclusions: This study provides generalizable evidence that ultra-sensitive discovery of TP53 mutations prior to cellular therapy is possible and could be a potentially valuable clinical tool to screen for tMN risk.