Deep targeted sequencing technologies are still not widely used in clinical practice due to the complexity of the methods and their cost. The Molecular Inversion Probes (MIP) technology is cost effective and scalable in the number of targets, however, suffers from low overall performance especially in GC rich regions. In order to improve the MIP performance, we sequenced a large cohort of healthy individuals (n = 4417), with a panel of 616 MIPs, at high depth in duplicates. To improve the previous state-of-the-art statistical model for low variant allele frequency, we selected 4635 potentially positive variants and validated them using amplicon sequencing. Using machine learning prediction tools, we significantly improved precision of 10–56.25% (P < 0.0004) to detect variants with VAF > 0.005. We further developed biochemically modified MIP protocol and improved its turn-around-time to ∼4 h. Our new biochemistry significantly improved uniformity, GC-Rich regions coverage, and enabled 95% on target reads in a large MIP panel of 8349 genomic targets. Overall, we demonstrate an enhancement of the MIP targeted sequencing approach in both detection of low frequency variants and in other key parameters, paving its way to become an ultrafast cost-effective research and clinical diagnostic tool.

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemo-resistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPCs) and lead to chemo-resistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPCs. Intriguingly, a misfolded P53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between wild-type (WT) and “pseudo-mutant” conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPCs from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPCs the pseudomutant conformation is the dominant. HSPCs from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPCs with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3AR882H-mutated HSPCs, suggesting that while a pre-leukemic mutation can predispose for P53 misfolding, additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53, specifically eliminated preL-HSPCs that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preLHSPCs carrying DNMT3A mutations. This opens new avenues for leukemia prevention.

No abstract

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemo-resistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPCs) and lead to chemo-resistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPCs. Intriguingly, a misfolded p53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between a wild-type (WT) and a pseudo-mutant conformations of p53. By combining single cell analyses and p53 conformation-specific monoclonal antibodies we studied preL-HSPCs from primary human DNMT3A AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPCs the pseudo-mutant conformation is the dominant. HSPCs from non-leukemic samples expressed both conformations to a similar extent. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of p53, specifically eliminated preL-HSPCs that had dysfunctional canonical p53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable p53 dysfunction in human preL-HSPCs carrying DNMT3A mutations. This opens new avenues for leukemia prevention.

Donor clonal hematopoiesis may be transferred to the recipient through allogeneic hematopoietic stem cell transplantation (HCT), but the potential for adverse long-term impact on transplant outcomes remains unknown. A total of 744 samples from 372 recipients who received HCT and the corresponding donors were included. Bar-coded error-corrected sequencing using a modified molecular inversion probe capture protocol was performed, which targeted 34 genes covering mutations involved in clonal hematopoiesis with indeterminate potential (CHIP) and other AML-related mutations. A total of 30 mutations were detected from 25 donors (6.7%): the most frequently mutated gene was TET2 (n=7, 28%), followed by DNMT3A (n=4, 16%), SMC3 (n=3, 12%) and SF3B1 (n=3, 12%). With a median follow-up duration of 13 years among survivors, the presence of CHIP in the donor was not associated with recipient overall survival (p=0.969), relapse incidence (p=0.600) or non-relapse mortality (p=0.570). Donor CHIP did not impair neutrophil (p=0.460) or platelet (p=0.250) engraftment, the rates of acute (p=0.490), or chronic graft-vs-host disease (p=0.220). No significant difference was noted for secondary malignancy following HCT between the two groups. The present study suggests that the presence of CHIP in allogeneic stem donors does not adversely affect transplant outcomes after HCT. Accordingly, further study is warranted to reach a clearer conclusion on whether molecular profiling to determine the presence of CHIP mutations is necessary for the pre-transplant evaluation of donors prior to stem cell donation.