Abstract
Introduction:TP53 mutated myeloid neoplasms (MNs) are associated with aberrant copy number variations (CNVs), complex karyotype (CK) and dismal outcomes. Recent studies showed that TP53 mutated clonal hematopoiesis (CH) can also harbor subclonal populations with CNVs; and is linked with higher risk of transformation into therapy-related myeloid neoplasm (tMN) following chemotherapy. To track the evolution of CK myeloid entities and the role TP53 mutations play in disease development, we integrated single-cell whole genome sequencing (scWGS-seq) by direct library preparation (DLP, Laks et al 2019) and transcriptome sequencing (scRNA-seq) to track the clonal dynamics of CH, MN and tMN.
Methods: 28 samples from 20 patients harboring TP53 mutations were ascertained by MSK-IMPACT panel (VAF >2%): 5 samples from 5 CH patients, 2 from 1 CCUS patient, 12 from 8 tMN patients, and 9 from 6 MN patients. scWGS-seq was performed using DLP, which offers single cell detection of CNV, single nucleotide variants (SNV), mutational signatures, and structural variants (SV). scRNA-seq was done using the 10x Genomics Chromium Single Cell 5' system; CNVs were inferred using InferCNV and Numbat packages. Cells with ≥3 CNVs were classified as CK.
Results: We generated scRNA and scWGS data for 218280 cells from 28 samples and 8154 cells from 8 samples, respectively. In CH/CCUS, no CNVs were identified by scRNA (Total cells: 60253) or DLP (Total cells: 1524), despite high TP53 VAF (median of 24%, range: 11%-49%) and detection of TP53 mutations by both methods. In contrast, CNVs were detected in all 21 of MN/tMN samples by scRNA (median TP53 VAF=43% with range 4%-91%), and in 19/21 samples (13/14 patients) these CNVs presented as CK. In 7 CK samples with both scRNA and DLP, the latter provided higher resolution and revealed subclonal branching.
In all 13 patients with CK clones, we noted 3 distinct modes of clonal evolution; single dominant clone (n=6), linear (n=4) and parallel (n=3) multi-clonal progressions, in line with Leppä at el 2024. Bi-allelic TP53 hits were detected in all CK clones. Importantly, in 3/5 patients with DLP, TP53-mutant cells lacking CK were identified, representing the most recent common ancestor predating CNV (del5q/del7q) acquisitions. In one patient with a history of exposure to platinum, platinum-associated mutational signatures (SBS31) was only observed in the CK branches of the phylogenetic tree, indicating that CNV and CK clones developed from a TP53 mutated CH clone already presented at the time of platinum exposure. Together with the absence of CNV in CH/CCUS, these findings showed that TP53 mutations always precede CK acquisitions (del5q/del7q) in myeloid pathogenesis.
Thanks to DLP resolution, we identified partially overlapping chromothripsis in 4 tMN patients, resulting in focal hyperamplification on chr19p. This stratified CK into clones with chr19p hypergain (CK-19p) and without (CK). To validate this finding, we examined 88 TP53-mutant CK MN and tMN patients in the MSK-IMPACT database, and identified 9 (10%) with chr19p hypergain events. In all 9 cases, CK-19p clones appeared only after therapy, suggesting that chr19p hypergain was a late, therapy selected event.
Transcriptomic profiling of clones' phenotype with scRNA revealed a bias toward progenitor cells in CK clones (42% vs 2% in non-CK clones) and MkEP-Erythroblasts in CK-19p clones (96% vs 4% in non-CK clones). Differential gene expression analysis showed that 1) CK progenitor clones were less proliferative with LSC quiescence-like phenotype (Zeng et al. 2022) and lower MYBL2 expression vs non-CK clones; 2) CK-19p MkEP-Erythroblasts clones were more proliferative than CK progenitor clones. These findings suggest that LSC quiescence-like phenotype is a hallmark of CK clones; and chr19p hypergain drives proliferation, resistance and a potentially distinct entity.
Conclusions: Using scRNA and scWGS-seq, we showed that biallelic TP53 inactivation is the founding event driving diverse CK clones in MN and tMN patients. In contrast, in tMN, chr19 hypergain emerged as a late event promoting a proliferative, MkEP-Erythroid phenotype, potentially as a novel mechanism for resistance and disease evolution. Finally, we identified LSC quiescence-like phenotype and lower MYBL2 expression as the hallmarks of CK clones, enabling survival and evolution under cytotoxic pressure.
