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Nature Biotechnology Jul 2022The combination of single-cell transcriptomics with mitochondrial DNA variant detection can be used to establish lineage relationships in primary human cells, but...
The combination of single-cell transcriptomics with mitochondrial DNA variant detection can be used to establish lineage relationships in primary human cells, but current methods are not scalable to interrogate complex tissues. Here, we combine common 3' single-cell RNA-sequencing protocols with mitochondrial transcriptome enrichment to increase coverage by more than 50-fold, enabling high-confidence mutation detection. The method successfully identifies skewed immune-cell expansions in primary human clonal hematopoiesis.
Topics: DNA, Mitochondrial; High-Throughput Nucleotide Sequencing; Humans; Mitochondria; Mutation; Sequence Analysis, RNA; Single-Cell Analysis
PubMed: 35210612
DOI: 10.1038/s41587-022-01210-8 -
Nature Medicine Dec 2019Accurate identification of tumor-derived somatic variants in plasma circulating cell-free DNA (cfDNA) requires understanding of the various biological compartments... (Clinical Trial)
Clinical Trial
Accurate identification of tumor-derived somatic variants in plasma circulating cell-free DNA (cfDNA) requires understanding of the various biological compartments contributing to the cfDNA pool. We sought to define the technical feasibility of a high-intensity sequencing assay of cfDNA and matched white blood cell DNA covering a large genomic region (508 genes; 2 megabases; >60,000× raw depth) in a prospective study of 124 patients with metastatic cancer, with contemporaneous matched tumor tissue biopsies, and 47 controls without cancer. The assay displayed high sensitivity and specificity, allowing for de novo detection of tumor-derived mutations and inference of tumor mutational burden, microsatellite instability, mutational signatures and sources of somatic mutations identified in cfDNA. The vast majority of cfDNA mutations (81.6% in controls and 53.2% in patients with cancer) had features consistent with clonal hematopoiesis. This cfDNA sequencing approach revealed that clonal hematopoiesis constitutes a pervasive biological phenomenon, emphasizing the importance of matched cfDNA-white blood cell sequencing for accurate variant interpretation.
Topics: Adult; Biomarkers, Tumor; Cell-Free Nucleic Acids; Circulating Tumor DNA; DNA Mutational Analysis; DNA, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Genomics; High-Throughput Nucleotide Sequencing; Humans; Male; Microsatellite Instability; Middle Aged; Mutation; Neoplasms
PubMed: 31768066
DOI: 10.1038/s41591-019-0652-7 -
Leukemia Oct 2022Nucleophosmin 1 (NPM1) is a nucleus-cytoplasmic shuttling protein which is predominantly located in the nucleolus and exerts multiple functions, including regulation of... (Review)
Review
Nucleophosmin 1 (NPM1) is a nucleus-cytoplasmic shuttling protein which is predominantly located in the nucleolus and exerts multiple functions, including regulation of centrosome duplication, ribosome biogenesis and export, histone assembly, maintenance of genomic stability and response to nucleolar stress. NPM1 mutations are the most common genetic alteration in acute myeloid leukemia (AML), detected in about 30-35% of adult AML and more than 50% of AML with normal karyotype. Because of its peculiar molecular and clinico-pathological features, including aberrant cytoplasmic dislocation of the NPM1 mutant and wild-type proteins, lack of involvement in driving clonal hematopoiesis, mutual exclusion with recurrent cytogenetic abnormalities, association with unique gene expression and micro-RNA profiles and high stability at relapse, NPM1-mutated AML is regarded as a distinct genetic entity in the World Health Organization (WHO) classification of hematopoietic malignancies. Starting from the structure and functions of NPM1, we provide an overview of the potential targeted therapies against NPM1-mutated AML and discuss strategies aimed at interfering with the oligomerization (compound NSC348884) and the abnormal traffic of NPM1 (avrainvillamide, XPO1 inhibitors) as well as at inducing selective NPM1-mutant protein degradation (ATRA/ATO, deguelin, (-)-epigallocatechin-3-gallate, imidazoquinoxaline derivatives) and at targeting the integrity of nucleolar structure (actinomycin D). We also discuss the current therapeutic results obtained in NPM1-mutated AML with the BCL-2 inhibitor venetoclax and the preliminary clinical results using menin inhibitors targeting HOX/MEIS1 expression. Finally, we review various immunotherapeutic approaches in NPM1-mutated AML, including immune check-point inhibitors, CAR and TCR T-cell-based therapies against neoantigens created by the NPM1 mutations.
Topics: Adult; Dactinomycin; Histones; Humans; Leukemia, Myeloid, Acute; Mutation; Nuclear Proteins; Nucleophosmin; Proto-Oncogene Proteins c-bcl-2; RNA; Receptors, Antigen, T-Cell
PubMed: 36008542
DOI: 10.1038/s41375-022-01666-2 -
Nature Medicine Nov 2021Clonal hematopoiesis (CH) results from somatic genomic alterations that drive clonal expansion of blood cells. Somatic gene mutations associated with hematologic...
Clonal hematopoiesis (CH) results from somatic genomic alterations that drive clonal expansion of blood cells. Somatic gene mutations associated with hematologic malignancies detected in hematopoietic cells of healthy individuals, referred to as CH of indeterminate potential (CHIP), have been associated with myeloid malignancies, while mosaic chromosomal alterations (mCAs) have been associated with lymphoid malignancies. Here, we analyzed CHIP in 55,383 individuals and autosomal mCAs in 420,969 individuals with no history of hematologic malignancies in the UK Biobank and Mass General Brigham Biobank. We distinguished myeloid and lymphoid somatic gene mutations, as well as myeloid and lymphoid mCAs, and found both to be associated with risk of lineage-specific hematologic malignancies. Further, we performed an integrated analysis of somatic alterations with peripheral blood count parameters to stratify the risk of incident myeloid and lymphoid malignancies. These genetic alterations can be readily detected in clinical sequencing panels and used with blood count parameters to identify individuals at high risk of developing hematologic malignancies.
Topics: Blood Cell Count; Chromosome Aberrations; Clonal Hematopoiesis; Hematologic Neoplasms; Hematopoietic Stem Cells; Humans
PubMed: 34663986
DOI: 10.1038/s41591-021-01521-4 -
Seminars in Hematology Apr 2019Acute myeloid leukemia (AML) was one of the first cancers to be sequenced at the level of the whole genome. Molecular profiling of AML through targeted sequencing panels... (Review)
Review
Acute myeloid leukemia (AML) was one of the first cancers to be sequenced at the level of the whole genome. Molecular profiling of AML through targeted sequencing panels and cytogenetics has become a mainstay in risk-stratifying AML patients and guiding clinicians toward optimal therapies for their patients. The extensive high-resolution genomic data generated to characterize AML have been instrumental in revealing the tremendous biological complexity of the disease, dictated in part by mutational, clonal, and epigenetic heterogeneity. This is further complicated by the antecedent nonleukemic state of clonal hematopoiesis that nevertheless is associated with an increased risk of developing a hematologic malignancy and with a greater risk of mortality from ischemic cardiovascular disease. Here in this review, we discuss developments in the field of AML biology and therapeutics, with a focus on advances in our understanding of how genetic and epigenetic determinants of AML have influenced prognostication and recent shifts in treatment paradigms, particularly within the context of precision oncology, for this highly complex group of hematologic malignancies.
Topics: Epigenesis, Genetic; Epigenomics; Humans; Leukemia, Myeloid, Acute
PubMed: 30926095
DOI: 10.1053/j.seminhematol.2018.08.001 -
Nature Medicine Oct 2021Germline SAMD9 and SAMD9L mutations (SAMD9/9L) predispose to myelodysplastic syndromes (MDS) with propensity for somatic rescue. In this study, we investigated a...
Germline SAMD9 and SAMD9L mutations (SAMD9/9L) predispose to myelodysplastic syndromes (MDS) with propensity for somatic rescue. In this study, we investigated a clinically annotated pediatric MDS cohort (n = 669) to define the prevalence, genetic landscape, phenotype, therapy outcome and clonal architecture of SAMD9/9L syndromes. In consecutively diagnosed MDS, germline SAMD9/9L accounted for 8% and were mutually exclusive with GATA2 mutations present in 7% of the cohort. Among SAMD9/9L cases, refractory cytopenia was the most prevalent MDS subtype (90%); acquired monosomy 7 was present in 38%; constitutional abnormalities were noted in 57%; and immune dysfunction was present in 28%. The clinical outcome was independent of germline mutations. In total, 67 patients had 58 distinct germline SAMD9/9L clustering to protein middle regions. Despite inconclusive in silico prediction, 94% of SAMD9/9L suppressed HEK293 cell growth, and mutations expressed in CD34 cells induced overt cell death. Furthermore, we found that 61% of SAMD9/9L patients underwent somatic genetic rescue (SGR) resulting in clonal hematopoiesis, of which 95% was maladaptive (monosomy 7 ± cancer mutations), and 51% had adaptive nature (revertant UPD7q, somatic SAMD9/9L). Finally, bone marrow single-cell DNA sequencing revealed multiple competing SGR events in individual patients. Our findings demonstrate that SGR is common in SAMD9/9L MDS and exemplify the exceptional plasticity of hematopoiesis in children.
Topics: Adolescent; Bone Marrow Cells; Child; Child, Preschool; Clonal Evolution; Clonal Hematopoiesis; Female; GATA2 Transcription Factor; Germ-Line Mutation; HEK293 Cells; High-Throughput Nucleotide Sequencing; Humans; Infant; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Male; Myelodysplastic Syndromes; Single-Cell Analysis; Tumor Suppressor Proteins
PubMed: 34621053
DOI: 10.1038/s41591-021-01511-6 -
Pharmacology & Therapeutics Mar 2020Circulating tumor DNA holds substantial promise as an early detection biomarker, particularly for cancers that do not have currently accepted screening methodologies,... (Review)
Review
Circulating tumor DNA holds substantial promise as an early detection biomarker, particularly for cancers that do not have currently accepted screening methodologies, such as ovarian, pancreatic, and gastric cancers. Many features intrinsic to ctDNA analysis may be leveraged to enhance its use as an early cancer detection biomarker: including ctDNA fragment lengths, DNA copy number variations, and associated patient phenotypic information. Furthermore, ctDNA testing may be synergistically used with other multi-omic biomarkers to enhance early detection. For instance, assays may incorporate early detection proteins (i.e., CA-125), epigenetic markers, circulating tumor RNA, nucleosomes, exosomes, and associated immune markers. Many companies are currently competing to develop a marketable early cancer detection test that leverages ctDNA. Although some hurdles (like early stage disease assay accuracy, high implementation costs, confounding from clonal hematopoiesis, and lack of clinical utility studies) need to be addressed before integration into healthcare, ctDNA assays hold substantial potential as an early cancer screening test.
Topics: Animals; Circulating Tumor DNA; Early Detection of Cancer; Humans; Neoplasms
PubMed: 31863816
DOI: 10.1016/j.pharmthera.2019.107458 -
Cancer Discovery Jan 2022Clonal hematopoiesis is a prevalent age-related condition associated with a greatly increased risk of hematologic disease; mutations in DNA methyltransferase 3A () are...
Clonal hematopoiesis is a prevalent age-related condition associated with a greatly increased risk of hematologic disease; mutations in DNA methyltransferase 3A () are the most common driver of this state. variants occur across the gene with some particularly associated with malignancy, but the functional relevance and mechanisms of pathogenesis of the majority of mutations are unknown. Here, we systematically investigated the methyltransferase activity and protein stability of 253 disease-associated mutations, and found that 74% were loss-of-function mutations. Half of these variants exhibited reduced protein stability and, as a class, correlated with greater clonal expansion and acute myeloid leukemia development. We investigated the mechanisms underlying the instability using a CRISPR screen and uncovered regulated destruction of DNMT3A mediated by the DCAF8 E3 ubiquitin ligase adaptor. We establish a new paradigm to classify novel variants that has prognostic and potential therapeutic significance for patients with hematologic disease. SIGNIFICANCE: DNMT3A has emerged as the most important epigenetic regulator and tumor suppressor in the hematopoietic system. Our study represents a systematic and high-throughput method to characterize the molecular impact of missense mutations and the discovery of a regulated destruction mechanism of DNMT3A offering new prognostic and future therapeutic avenues...
Topics: Animals; DNA Methyltransferase 3A; HEK293 Cells; Humans; Leukemia, Myeloid, Acute; Leukocytes, Mononuclear; Mice; Mutation, Missense; Ubiquitin-Protein Ligases
PubMed: 34429321
DOI: 10.1158/2159-8290.CD-21-0560 -
Nature Oct 2020Age is the dominant risk factor for most chronic human diseases, but the mechanisms through which ageing confers this risk are largely unknown. The age-related...
Age is the dominant risk factor for most chronic human diseases, but the mechanisms through which ageing confers this risk are largely unknown. The age-related acquisition of somatic mutations that lead to clonal expansion in regenerating haematopoietic stem cell populations has recently been associated with both haematological cancer and coronary heart disease-this phenomenon is termed clonal haematopoiesis of indeterminate potential (CHIP). Simultaneous analyses of germline and somatic whole-genome sequences provide the opportunity to identify root causes of CHIP. Here we analyse high-coverage whole-genome sequences from 97,691 participants of diverse ancestries in the National Heart, Lung, and Blood Institute Trans-omics for Precision Medicine (TOPMed) programme, and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid and inflammatory traits that are specific to different CHIP driver genes. Association of a genome-wide set of germline genetic variants enabled the identification of three genetic loci associated with CHIP status, including one locus at TET2 that was specific to individuals of African ancestry. In silico-informed in vitro evaluation of the TET2 germline locus enabled the identification of a causal variant that disrupts a TET2 distal enhancer, resulting in increased self-renewal of haematopoietic stem cells. Overall, we observe that germline genetic variation shapes haematopoietic stem cell function, leading to CHIP through mechanisms that are specific to clonal haematopoiesis as well as shared mechanisms that lead to somatic mutations across tissues.
Topics: Adult; Africa; Aged; Aged, 80 and over; Black People; Cell Self Renewal; Clonal Hematopoiesis; DNA-Binding Proteins; Dioxygenases; Female; Genetic Predisposition to Disease; Genome, Human; Germ-Line Mutation; Hematopoietic Stem Cells; Humans; Intracellular Signaling Peptides and Proteins; Male; Middle Aged; National Heart, Lung, and Blood Institute (U.S.); Phenotype; Precision Medicine; Proto-Oncogene Proteins; Tripartite Motif Proteins; United States; Whole Genome Sequencing; alpha Karyopherins
PubMed: 33057201
DOI: 10.1038/s41586-020-2819-2 -
Nature Jun 2022Age-related change in human haematopoiesis causes reduced regenerative capacity, cytopenias, immune dysfunction and increased risk of blood cancer, but the reason for...
Age-related change in human haematopoiesis causes reduced regenerative capacity, cytopenias, immune dysfunction and increased risk of blood cancer, but the reason for such abrupt functional decline after 70 years of age remains unclear. Here we sequenced 3,579 genomes from single cell-derived colonies of haematopoietic cells across 10 human subjects from 0 to 81 years of age. Haematopoietic stem cells or multipotent progenitors (HSC/MPPs) accumulated a mean of 17 mutations per year after birth and lost 30 base pairs per year of telomere length. Haematopoiesis in adults less than 65 years of age was massively polyclonal, with high clonal diversity and a stable population of 20,000-200,000 HSC/MPPs contributing evenly to blood production. By contrast, haematopoiesis in individuals aged over 75 showed profoundly decreased clonal diversity. In each of the older subjects, 30-60% of haematopoiesis was accounted for by 12-18 independent clones, each contributing 1-34% of blood production. Most clones had begun their expansion before the subject was 40 years old, but only 22% had known driver mutations. Genome-wide selection analysis estimated that between 1 in 34 and 1 in 12 non-synonymous mutations were drivers, accruing at constant rates throughout life, affecting more genes than identified in blood cancers. Loss of the Y chromosome conferred selective benefits in males. Simulations of haematopoiesis, with constant stem cell population size and constant acquisition of driver mutations conferring moderate fitness benefits, entirely explained the abrupt change in clonal structure in the elderly. Rapidly decreasing clonal diversity is a universal feature of haematopoiesis in aged humans, underpinned by pervasive positive selection acting on many more genes than currently identified.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Child; Child, Preschool; Clonal Hematopoiesis; Clone Cells; Female; Hematologic Neoplasms; Hematopoietic Stem Cells; Humans; Infant; Infant, Newborn; Longevity; Male; Middle Aged; Multipotent Stem Cells; Young Adult
PubMed: 35650442
DOI: 10.1038/s41586-022-04786-y