Single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) resolves the heterogeneity of epigenetic states across cells but does not typically capture exonic mutations, which ...limits our knowledge of how somatic mutations alter chromatin landscapes. Here, we present a plate-based approach coupling high-sensitivity genotyping of genomic loci with high-content scATAC-seq libraries from the same single cells. We first describe steps for optimization of genotyping primers, followed by detailed guidance on the preparation of both scATAC-seq and single-cell genotyping libraries, fully automated on high-throughput liquid handling platforms.
For complete details on the use and execution of this protocol, please refer to Turkalj, Jakobsen et al.1
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•scATAC-seq coupled with high-sensitivity mutation capture at multiple genomic loci•Link genetic and epigenetic evolution in malignant and pre-malignant tissues•Guidance on barcoding, automation, and liquid handling for high-throughput applications
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Single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) resolves the heterogeneity of epigenetic states across cells but does not typically capture exonic mutations, which limits our knowledge of how somatic mutations alter chromatin landscapes. Here, we present a plate-based approach coupling high-sensitivity genotyping of genomic loci with high-content scATAC-seq libraries from the same single cells. We first describe steps for optimization of genotyping primers, followed by detailed guidance on the preparation of both scATAC-seq and single-cell genotyping libraries, fully automated on high-throughput liquid handling platforms.
The haematological malignancies are a heterogeneous group of neoplastic disorders, which lead to almost 10,000 deaths annually in the UK. Over the past 2 decades, there has been significant progress ...in our understanding of the pathological mechanisms underlying these cancers, accompanied by improvements in outcomes for some patients. In particular, advances in next-generation sequencing now make it possible to define the genetic lesions present in each patient, which has led to improved disease classification, risk stratification and identification of new therapeutic targets. Here we discuss recent advances in the genomic classification and targeted treatment of haematological malignancies, focusing on acute myeloid leukaemia. Multiple novel drug classes are now on the horizon, including agents that target overactive signalling pathways, differentiation therapies and immunotherapies. By combining molecular diagnostics with targeted therapy, the management of these diseases is set to change radically over the coming years.
The haematological malignancies are a heterogeneous group of neoplastic disorders, which lead to almost 10,000 deaths annually in the UK. Over the past 2 decades, there has been significant progress ...in our understanding of the pathological mechanisms underlying these cancers, accompanied by improvements in outcomes for some patients. In particular, advances in next-generation sequencing now make it possible to define the genetic lesions present in each patient, which has led to improved disease classification, risk stratification and identification of new therapeutic targets. Here we discuss recent advances in the genomic classification and targeted treatment of haematological malignancies, focusing on acute myeloid leukaemia. Multiple novel drug classes are now on the horizon, including agents that target overactive signalling pathways, differentiation therapies and immunotherapies. By combining molecular diagnostics with targeted therapy, the management of these diseases is set to change radically over the coming years.
•Fractionated compared with single-dose gemtuzumab increased response depth across most AML molecular groups without increasing toxicity.•Older patients with AML receiving fractionated gemtuzumab had ...improved survival when induction was consolidated by allograft.
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Addition of gemtuzumab ozogamicin (GO) to induction chemotherapy improves outcomes in older patients with acute myeloid leukemia (AML), but it is uncertain whether a fractionated schedule provides additional benefit to a single dose. We randomized 852 older adults (median age, 68-years) with AML/high-risk myelodysplasia to GO on day 1 (GO1) or on days 1 and 4 (GO2) of course 1 induction. The median follow-up period was 50.2 months. Although complete remission (CR) rates after course 1 did not significantly differ between arms (GO2, 63%; GO1, 57%; odds ratio OR, 0.78; P = .08), there were significantly more patients who achieved CR with a measurable residual disease (MRD)<0.1% (50% vs 41%; OR, 0.72; P = .027). This differential MRD reduction with GO2 varied across molecular subtypes, being greatest for IDH mutations. The 5-year overall survival (OS) was 29% for patients in the GO2 arm and 24% for those in the GO1 arm (hazard ratio HR, 0.89; P = .14). In a sensitivity analysis excluding patients found to have adverse cytogenetics or TP53 mutations, the 5-year OS was 33% for GO2 and 26% for GO1 (HR, 0.83; P = .045). In total, 228 (27%) patients received an allogeneic transplantation in first remission. Posttransplant OS was superior in the GO2 arm (HR, 0.67; P = .033); furthermore, the survival advantage from GO2 in the sensitivity analysis was lost when data of patients were censored at transplantation. In conclusion, GO2 was associated with a greater reduction in MRD and improved survival in older adults with nonadverse risk genetics. This benefit from GO2 was dependent on allogeneic transplantation to translate the better leukemia clearance into improved survival. This trial was registered at www.isrctn.com as #ISRCTN 31682779.
Understanding clonal evolution and cancer development requires experimental approaches for characterizing the consequences of somatic mutations on gene regulation. However, no methods currently exist ...that efficiently link high-content chromatin accessibility with high-confidence genotyping in single cells. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), enabling accurate mutation detection at multiple amplified loci, coupled with robust chromatin accessibility readout. We applied GTAC to primary acute myeloid leukemia, obtaining high-quality chromatin accessibility profiles and clonal identities for multiple mutations in 88% of cells. We traced chromatin variation throughout clonal evolution, showing the restriction of different clones to distinct differentiation stages. Furthermore, we identified switches in transcription factor motif accessibility associated with a specific combination of driver mutations, which biased transformed progenitors toward a leukemia stem cell-like chromatin state. GTAC is a powerful tool to study clonal heterogeneity across a wide spectrum of pre-malignant and neoplastic conditions.
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•High-sensitivity genomic genotyping linked to scATAC-seq in single malignant cells•Characterization of epigenetic variation as preleukemic clones transform to leukemia•Differentiation of specific leukemic clones is delayed at an LSC-like chromatin state•Genomic elements perturbed in LSC-like clones relate to stemness and differentiation
Turkalj, Jakobsen, et al. develop a method linking clonal identities of single cells to their chromatin accessibility and study how epigenetic landscapes change with clonal evolution in human acute myeloid leukemia, revealing altered accessibility of genes and transcription factor motifs linked to leukemia stemness downstream of cooperating somatic mutations.
Clonal hematopoiesis (CH) arises when hematopoietic stem cells (HSCs) acquire mutations, most frequently in the DNMT3A and TET2 genes, conferring a competitive advantage through mechanisms that ...remain unclear. To gain insight into how CH mutations enable gradual clonal expansion, we used single-cell multi-omics with high-fidelity genotyping on human CH bone marrow (BM) samples. Most of the selective advantage of mutant cells occurs within HSCs. DNMT3A- and TET2-mutant clones expand further in early progenitors, while TET2 mutations accelerate myeloid maturation in a dose-dependent manner. Unexpectedly, both mutant and non-mutant HSCs from CH samples are enriched for inflammatory and aging transcriptomic signatures, compared with HSCs from non-CH samples, revealing a non-cell-autonomous effect. However, DNMT3A- and TET2-mutant HSCs have an attenuated inflammatory response relative to wild-type HSCs within the same sample. Our data support a model whereby CH clones are gradually selected because they are resistant to the deleterious impact of inflammation and aging.
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•High-fidelity single-cell genotyping and transcriptomes of human clonal hematopoiesis•Most clonal expansion occurs in HSCs; TET2MUT clones also have a myeloid bias•HSCs from CH samples show increased inflammation and aging-related gene expression•These deleterious effects are attenuated in DNMT3AMUT and TET2MUT HSCs
Vyas and colleagues show DNMT3A and TET2 mutation-specific patterns of clonal expansion in human clonal hematopoiesis (CH). Hematopoietic stem cells (HSCs) in CH bone marrow have heightened transcriptional responses to inflammation. However, these responses are attenuated in CH mutant HSCs, and this could contribute to their clonal advantage.
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