Perturbed hematopoiesis in individuals with germline DNMT3A overgrowth Tatton-Brown-Rahman syndrome

Tatton-Brown-Rahman syndrome (TBRS) is an overgrowth disorder caused by germline heterozygous mutations in the DNA methyltransferase DNMT3A. DNMT3A is a critical regulator of hematopoietic stem cell (HSC) differentiation and somatic DNMT3A mutations are frequent in hematologic malignancies and clonal hematopoiesis. Yet, the impact of constitutive DNMT3A mutation on hematopoiesis in TBRS is undefined. In order to establish how constitutive mutation of DNMT3A impacts blood development in TBRS we gathered clinical data and analyzed blood parameters in 18 individuals with TBRS. We also determined the distribution of major peripheral blood cell lineages by flow cytometric analyses. Our analyses revealed non-anemic macrocytosis, a relative decrease in lymphocytes and increase in neutrophils in TBRS individuals compared to unaffected controls. We were able to recapitulate these hematologic phenotypes in multiple murine models of TBRS and identified rare hematological and non-hematological malignancies associated with constitutive Dnmt3a mutation. We further show that loss of DNMT3A in TBRS is associated with an altered DNA methylation landscape in hematopoietic cells affecting regions critical to stem cell function and tumorigenesis. Overall, our data identify key hematopoietic effects driven by DNMT3A mutation with clinical implications for individuals with TBRS and DNMT3A-associated clonal hematopoiesis or malignancies.     

Utility of plasma cell-free DNA for de novo detection and quantification of clonal hematopoiesis

Although cell-free DNA (cfDNA) tests have emerged as a potential non-invasive alternative to bone marrow biopsies for monitoring clonal hematopoiesis in hematologic diseases, whether commercial cfDNA assays can be implemented for the detection and quantification of de novo clonal hematopoiesis in place of blood cells is uncertain. In this study, peripheral plasma cfDNA samples available from patients with aplastic anemia (n=25) or myelodysplastic syndromes (n=27) and a healthy cohort (n=107) were screened for somatic variants in genes related to hematologic malignancies using a Clinical Laboratory Improvement Amendments-certified panel. Results were further compared to DNA sequencing of matched blood cells. In reported results, 85% of healthy subjects, 36% of patients with aplastic anemia and 74% of patients with myelodysplastic syndromes were found to have somatic cfDNA variants, most frequently in DNMT3A, TET2, ASXL1 and SF3B1. However, concordance between cfDNA and blood cell findings was poor for the detection of clonal hematopoiesis when the allele frequency of the variants was <10%, which was mostly observed in the healthy and aplastic anemia cohorts but not in patients with myelodysplastic syndromes. After filtering data for potential artifacts due to low variant allele frequency and sequencing depth, the frequency of clonal hematopoiesis in cfDNA from healthy individuals and patients with aplastic anemia decreased to 52% and 20%, respectively. cfDNA and matched blood cells were not interchangeable for tracking changes in allele burdens as their agreement by Bland-Altman analysis was poor. A commercial cfDNA assay had good performance for de novo detection of clonal hematopoiesis in myelodysplastic syndromes, but showed no advantage over blood cells in diseases with low allele burdens or in healthy individuals.     

Clonal Hematopoiesis and therapy related MDS/AML

Clonal Hematopoiesis is defined as the presence of mutations in peripheral blood in the absence of myeloid malignancies and is thought to occur as a normal part of ageing due to the fitness advantage conferred by these mutations in an ageing hematopoietic compartment. Therapy related myeloid neoplasms are malignancies that occur after exposure to chemotherapy/radiation and are associated with poor survival. Clonal hematopoiesis mutations represent a pre malignant state that can be triggered by exposure to cytotoxic damage and rapid hematopoietic stem cell expansion. We discuss in this review clinical evidence of association of clonal hematopoiesis with risk of therapy related myeloid neoplasms, the underlying mechanisms of clonal expansion under different cellular stresses and recommendations on clinical follow up of patients with clonal hematopoiesis including possible strategies for prevention of therapy related myeloid neoplasms.     

Clonal hematopoiesis in patients with dyskeratosis congenita

Dyskeratosis congenita (DC) is a rare inherited telomeropathy most frequently caused by mutations in a number of genes all thought to be involved in telomere maintenance. The main causes of mortality in DC are bone marrow failure as well as malignancies including leukemias and solid tumors. The clinical picture including the degree of bone marrow failure is highly variable and factors that contribute to this variability are poorly understood. Based on the recent finding of frequent clonal hematopoiesis in related bone marrow failure syndromes, we hypothesized that somatic mutations may also occur in DC and may contribute at least in part to the variability in blood production. To evaluate for the presence of clonal hematopoiesis in DC, we used a combination of X‐inactivation, comparative whole exome sequencing (WES) and single nucleotide polymorphism array (SNP‐A) analyses. We found that clonal hematopoiesis in DC is common, as suggested by skewed X‐inactivation in 8 out of 9 female patients compared to 3 out of 10 controls, and by the finding of acquired copy neutral loss‐of‐heterozygosity on SNP‐A analysis. In addition, 3 out of 6 independent DC patients were found to have acquired somatic changes in their bone marrow by WES, including a somatic reversion in DKC1, as well as missense mutations in other protein coding genes. Our results indicate that clonal hematopoiesis is a common feature of DC, and suggest that such somatic changes, though commonly expected to indicate malignancy, may lead to improved blood cell production or stem cell survival. Am. J. Hematol. 91:1227–1233, 2016. © 2016 Wiley Periodicals, Inc.     

Acquired alpha-thalassemia in association with myelodysplastic syndrome and other hematologic malignancies

Abnormalities of hemoglobin synthesis are usually inherited but may also arise as a secondary manifestation of another disease, most commonly hematologic neoplasia. Acquired hemoglobin disorders can be seen in any population and are not restricted to areas of the world with high incidences of inherited hemoglobinopathies. In fact, the acquired hemoglobinopathies may be more readily recognized where inherited hemoglobin abnormalities are rare and less likely to cause diagnostic confusion. Acquired alpha-thalassemia is the best characterized of the acquired red blood cell disorders in patients with hematologic malignancy, and it is almost always associated with a myelodysplastic syndrome (MDS). At least 2 molecular mechanisms for acquired alpha-thalassemia are now recognized: acquired deletion of the alpha-globin gene cluster limited to the neoplastic clone and, more commonly, inactivating somatic mutations of the trans-acting chromatin-associated factor ATRX, which cause dramatic down-regulation of alpha-globin gene expression. Here we review the clinical, hematologic, and molecular genetic features of alpha-thalassemia arising in a clonal myeloid disorder, and we discuss howATRX might affect gene expression in normal and abnormal hematopoiesis through epigenetic mechanisms.     

Importance of clonal hematopoiesis in heart failure

Heart failure is prevalent in the elderly population. Inflammatory processes can contribute to the progression of heart failure by altering the balance of tissue healing and pathological remodeling during the injury response. New findings show that aging can alter immune cell phenotypes through the process of clonal hematopoiesis. This condition results from acquired somatic DNA mutations in specific driver genes that give rise to clonal expansions of mutant hematopoietic cells with overactive inflammatory properties. Recent clinical and experimental studies have shown that clonal hematopoiesis is prevalent in heart failure patients and associated with poor prognosis. In this review, we summarize current evidence that associates clonal hematopoiesis with the progression of heart failure. We further describe the mechanistic links between clonal hematopoiesis and the pro-inflammatory responses that can contribute to pathological outcomes in the heart. Finally, we provide perspectives on future research directions in the area of clonal hematopoiesis and heart failure.     

Genetic and epigenetic determinants of AML pathogenesis

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.     

Somatic,hematologic phenotype,long‐term outcome,and effect of hematopoietic stem cell transplantation. An analysis of 97 Fanconi anemia patients from the Italian national database on behalf of the Marrow Failure Study Group of the AIEOP (Italian Association of Pediatric Hematology–Oncology)

We analyzed 97 Fanconi anemia patients from a clinic/biological database for genotype, somatic, and hematologic phenotype, adverse hematological events, solid tumors, and treatment. Seventy‐two patients belonged to complementation group A. Eighty percent of patients presented with mild/moderate somatic phenotype and most with cytopenia. No correlation was seen between somatic/hematologic phenotype and number of missense mutations of FANCA alleles. Over follow‐up, 33% of patients improved or maintained mild/moderate cytopenia or normal blood count, whereas remaining worsened cytopenia. Eleven patients developed a hematological adverse event (MDS, AML, pathological cytogenetics) and three developed solid tumors. 10 years cumulative risk of death of the whole cohort was 25.6% with median follow‐up 5.8 years. In patients eligible to hematopoietic stem cell transplantation because of moderate cytopenia, mortality was significantly higher in subjects transplanted from matched unrelated donor over nontransplanted subjects, whereas there was no significant difference between matched sibling donor transplants and nontransplanted patients. In patients eligible to transplant because of severe cytopenia and clonal disease, mortality risk was not significantly different in transplanted from matched unrelated versus matched sibling donor versus nontransplanted subjects. The decision to transplant should rely on various elements including, type of donor, HLA matching, patient comorbidities, impairment, and clonal evolution of hematopoiesis. Am. J. Hematol. 91:666–671, 2016. © 2016 Wiley Periodicals, Inc.     

Clonal hematopoiesis and VEXAS syndrome: survival of the fittest clones?

Clonal hematopoiesis (CH) is defined by the acquisition of somatic mutations in hematopoietic stem cells (HSC) leading to enhanced cellular fitness and proliferation under positive clonal selection pressures. CH most frequently involves epigenetic regulator genes (DNMT3A, TET2 and ASXL1), with these mutations being associated with enhanced inflammation and increased all-cause mortality largely from cardiovascular disease and endothelial dysfunction. These mutations also increase the risk for hematological neoplasms. Somatic mutations in UBA1, encoding the E1 ubiquitin ligase in HSC, cause a severe adult-onset autoinflammatory disease that can be associated with myeloid and plasma cell neoplasms, termed VEXAS (vacuoles, X-linked, autoinflammatory, somatic) syndrome. Given the degree of inflammation seen, one would have expected this to be a fertile ground for CH development and propagation, however, preliminary data doesn't support this. Here in, we review the current data on CH, inflammation and VEXAS syndrome.     

Acquired somatic ATRX mutations in myelodysplastic syndrome associated with alpha thalassemia (ATMDS) convey a more severe hematologic phenotype than germline ATRX mutations

Acquired somatic mutations in ATRX, an X-linked gene encoding a chromatin-associated protein, were recently identified in 4 patients with the rare subtype of myelodysplastic syndrome (MDS) associated with thalassemia (ATMDS). Here we describe a series of novel point mutations in ATRX detected in archival DNA samples from marrow and/or blood of patients with ATMDS by use of denaturing high-performance liquid chromatography (DHPLC), a technique sensitive to low-level mosaicism. Two of the new mutations result in changes in amino acids altered in previously described pedigrees with germ line ATRX mutations (ATR-X syndrome), but the hematologic abnormalities were much more severe in the patients with ATMDS than in the corresponding constitutional cases. In one ATMDS case where DNA samples from several time points were available, the proportion of ATRX-mutant subclones correlated with changes in the amount of hemoglobin H. This study strengthens the link between acquired, somatic ATRX mutations and ATMDS, illustrates how molecular defects associated with MDS and other hematologic malignancies masked by somatic mosaicism may be detected by DHPLC, and shows that additional factors increase the severity of the hematologic phenotype of ATRX mutations in ATMDS.     

Juvenile myelomonocytic leukemia – A bona fide RASopathy syndrome

Juvenile myelomonocytic leukemia (JMML) is a pediatric myelodysplastic/myeloproliferative neoplasm overlap syndrome with sustained peripheral blood monocytosis, aggressive features, and poor outcomes. In >90% of cases JMML is driven by germline or somatic mutations involving the canonical RAS pathway (PTPN11, NRAS, CBL, KRAS and NF1), with somatic mutations/alterations in RAS pathway genes (second hit), SETBP1, ASXL1 and JAK3 resulting in disease progression. While spontaneous regression has been seen in germline PTPN11 and CBL mutant JMML, in most patients, allogeneic stem cell transplant is the only curative modality. JMML shares several phenotypic features with its adult counterpart proliferative, chronic myelomonocytic leukemia (pCMML). pCMML largely occurs due to RAS pathway mutations that occur in the context of age related clonal hematopoiesis (TET2, SRSF2, ASXL1), while JMML is a bona fide RASopathy, with additional somatic mutations, including in epigenetic regulators genes resulting in disease progression.     

Molecular pathogenesis of acquired aplastic anemia

The application of next‐generation sequencing (NGS) has enhanced our understanding of the genetic landscape in acquired aplastic anemia (AA). Parallel progress has been in addressing aspects underlying immune dysregulation in disease pathogenesis. Novel insights into the molecular and biologic mechanisms have led to a shift in the paradigm of AA, from a solely autoimmune pathogenic concept toward its recognition as a multifaceted pathophysiology characterized by cytogenetic abnormalities, recurrent somatic mutations, telomere attrition, and immune dysregulation. The detection of recurrent driver mutations disrupting myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML)‐associated genes has suggested a pathophysiologic link between clonal hematopoiesis in AA and the later development of these clonal disorders. Further, certain AA‐related somatic genetic alterations may have clinical implications on treatment response, disease progression, and survival following immunosuppressive therapy. Going forward, wider validation of these genetic abnormalities will allow for their incorporation into a more informative risk stratification system that does not rely solely on clinical factors.     

Tet2-Mediated Clonal Hematopoiesis Accelerates Heart Failure Through a Mechanism Involving the IL-1β/NLRP3 Inflammasome

Background

Recent studies have shown that hematopoietic stem cells can undergo clonal expansion secondary to somatic mutations in leukemia-related genes, thus leading to an age-dependent accumulation of mutant leukocytes in the blood. This somatic mutation-related clonal hematopoiesis is common in healthy older individuals, but it has been associated with an increased incidence of future cardiovascular disease. The epigenetic regulator TET2 is frequently mutated in blood cells of individuals exhibiting clonal hematopoiesis.

Genetic origins and clinical phenotype of familial and acquired erythrocytosis and thrombocytosis

Familial and acquired erythrocytosis and thrombocytosis are characterized by myeloid lineage hyperproliferation, which is either single or multi‐lineage in origin. The single lineage disorders exhibit Mendelian inheritance with polyclonal hematopoiesis and often arise from a single genetic defect. In contrast, the multi‐lineage disorders exhibit complex patterns of inheritance with multi‐genetic origins and clonal hematopoiesis. They have the potential to acquire JAK2 somatic mutations, but this is not the primary event. Identification of the disease‐causing genes will enable better classification of familial and acquired erythrocytosis and thrombocytosis. Furthermore, it will provide an insight into the mechanisms regulating myeloid cell proliferation. Am. J. Hematol., 2009. © 2008 Wiley‐Liss, Inc.     

Mast cell disease associated with acute myeloid leukemia: detection of a new c-kit mutation Asp816His

Mast cell disease (MCD), a proliferation of mast cells (MC), is occasionally associated with hematologic malignancies. Neoplastic MC have activating c-kit mutations. c-kit is a receptor tyrosine kinase required for the development, proliferation, and survival of MC. Interaction of c-kit with its ligand stem cell factor induces dimerization, receptor phosphorylation, and signal transduction. The most common c-kit mutation detected in neoplastic MCD is Asp816Val, which results in ligand-independent autophosphorylation of the receptor leading to MC proliferation. We describe the rare occurrence of MCD associated with acute myeloid leukemia, report a novel c-kit mutation Asp816 His, and discuss the pathogenesis of MCD associated with hematologic malignancies.     

L’hématopoïèse clonale : un concept émergent à la croisée des spécialités

Clonal hematopoiesis of undetermined significance or CHIP describes the identification, in individuals without hematologic disease, of one or more somatic mutations in hematopoietic cells. These mutations, detected by high-throughput genes sequencing (Next-Generation Sequencing or NGS), affect genes first identified in acute myeloid leukemia or myelodysplastic syndrome, such as DNMT3A, TET2 and ASXL1. CHIP is associated with an increased risk of malignant hemopathy, both myeloid and lymphoid, evaluated from 0.5 to 1% per year. CHIP is also associated with an increased risk of overall mortality and cardiovascular diseases. CHIP detection using NGS is currently limited to basic science field, but recent studies suggest that it may be of clinical interest.     

How predictive is the finding of clonal hematopoiesis for the development of myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML)?

Clonal hematopoiesis (CH) – a biological state in which one or a small number of hematopoietic stem or progenitor cells contribute disproportionately to blood cell production, usually as a result of somatic gene mutations in the stem cells – is often considered to be a precursor to myeloid neoplasia, especially myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). However, the majority of people with CH never develop an overt myeloid neoplasm, and CH can be a precursor to lymphoid cancers as well as myeloid neoplasms. In addition, CH increases all-cause mortality and augments the risk of several non-neoplastic medical conditions, including atherosclerotic cardiovascular disease. CH can arise during aging, or in the context of an inherited marrow failure syndrome, aplastic anemia, or hematopoietic cell transplantation. Risk factors for progression of CH to myeloid neoplasia include larger clone size; the presence of a TP53, IDH1/2, or splicing mutation; multiple mutations; and associated cytopenias or abnormal red blood cell indices. The receipt of genotoxic chemotherapy or radiation, which can promote clonal expansion of mutant clones at the expense of healthy progenitor cells, may result in therapy-related MDS/AML.