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Although aplastic anemia is responsive to immunosuppressive therapy, small subpopulations of hematopoietic cells with clonal gene mutations may exist, and different sets of mutations show distinct clinical behavior and response to therapy. Acquired aplastic anemia is caused by immune-mediated destruction of hematopoietic stem and progenitor cells. 1 CD34+ cells and early progenitors are uniformly reduced in aplastic anemia. 2 Bone marrow transplantation is curative, and patients may also have a response to immunosuppressive therapy. 3 , 4 With improved survival, the late development of myelodysplastic syndromes, acute myeloid leukemia (AML), or both has been noted in about 15% of patients and termed “clonal evolution.” 5 Although “clonal evolution” historically has been used to describe the development of cancer in patients with an immune disease, this term is a misnomer, since there is evidence of clonal hematopoiesis associated . . .

A randomized trial comparing immunosuppression (horse ATG and cyclosporine) with the same therapy plus eltrombopag showed more rapid and complete responses with the addition of eltrombopag. Severe adverse effects were similar in the two groups. Somatic mutations increased in both groups but did not lead to an increase in leukemia.

Cancer is a clonal disorder derived from a single ancestor cell and its progenies that are positively selected by acquisition of ‘driver mutations’. However, the evolution of positively selected clones does not necessarily imply the presence of cancer. On the contrary, it has become clear that expansion of these clones in phenotypically normal or non-cancer tissues is commonly seen in association with ageing and/or in response to environmental insults and chronic inflammation. Recent studies have reported expansion of clones harbouring mutations in cancer driver genes in the blood, skin, oesophagus, bronchus, liver, endometrium and bladder, where the expansion could be so extensive that tissues undergo remodelling of an almost entire tissue. The presence of common cancer driver mutations in normal tissues suggests a strong link to cancer development, providing an opportunity to understand early carcinogenic processes. Nevertheless, some driver mutations are unique to normal tissues or have a mutation frequency that is much higher in normal tissue than in cancer, indicating that the respective clones may not necessarily be destined for evolution to cancer but even negatively selected for carcinogenesis depending on the mutated gene. Moreover, tissues that are remodelled by genetically altered clones might define functionalities of aged tissues or modified inflammatory processes. In this Review, we provide an overview of major findings on clonal expansion in phenotypically normal or non-cancer tissues and discuss their biological significance not only in cancer development but also in ageing and inflammatory diseases.Clonal expansion in phenotypically normal or non-cancer tissues is commonly seen in association with ageing and/or in response to environmental insults and chronic inflammation, but does not necessarily indicate cancer development. This Review discusses recent findings on clonal expansion in these tissues and their biological significance in cancer development, ageing and inflammatory diseases.

There is much interest in the tissue of origin of circulating DNA in plasma. Data generated using DNA methylation markers have suggested that hematopoietic cells of white cell lineages are important contributors to the circulating DNA pool. However, it is not known whether cells of the erythroid lineage would also release DNA into the plasma. Using high-resolution methylation profiles of erythroblasts and other tissue types, 3 genomic loci were found to be hypomethylated in erythroblasts but hypermethylated in other cell types. We developed digital PCR assays for measuring erythroid DNA using the differentially methylated region for each locus. Based on the methylation marker in the ferrochelatase gene, erythroid DNA represented a median of 30.1% of the plasma DNA of healthy subjects. In subjects with anemia of different etiologies, quantitative analysis of circulating erythroid DNA could reflect the erythropoietic activity in the bone marrow. For patients with reduced erythropoietic activity, as exemplified by aplastic anemia, the percentage of circulating erythroid DNA was decreased. For patients with increased but ineffective erythropoiesis, as exemplified by β-thalassemia major, the percentage was increased. In addition, the plasma concentration of erythroid DNA was found to correlate with treatment response in aplastic anemia and iron deficiency anemia. Plasma DNA analysis using digital PCR assays targeting the other 2 differentially methylated regions showed similar findings. Erythroid DNA is a hitherto unrecognized major component of the circulating DNA pool and is a noninvasive biomarker for differential diagnosis and monitoring of anemia.

Eltrombopag, an FDA-approved non-peptidyl thrombopoietin receptor agonist, is clinically used for the treatment of aplastic anemia, a disease characterized by hematopoietic stem cell failure and pancytopenia, to improve platelet counts and stem cell function. Eltrombopag treatment results in a durable trilineage hematopoietic expansion in patients. Some of the eltrombopag hematopoietic activity has been attributed to its off-target effects, including iron chelation properties. However, the mechanism of action for its full spectrum of clinical effects is still poorly understood. Here, we report that eltrombopag bound to the TET2 catalytic domain and inhibited its dioxygenase activity, which was independent of its role as an iron chelator. The DNA demethylating enzyme TET2, essential for hematopoietic stem cell differentiation and lineage commitment, is frequently mutated in myeloid malignancies. Eltrombopag treatment expanded TET2-proficient normal hematopoietic stem and progenitor cells, in part because of its ability to mimic loss of TET2 with simultaneous thrombopoietin receptor activation. On the contrary, TET inhibition in TET2 mutant malignant myeloid cells prevented neoplastic clonal evolution in vitro and in vivo. This mechanism of action may offer a restorative therapeutic index and provide a scientific rationale to treat selected patients with TET2 mutant-associated or TET deficiency-associated myeloid malignancies.

Idiopathic aplastic anemia (IAA) pathophysiology is dominated by autoreactivity of human leukocyte antigen (HLA)-restricted T-cells against antigens presented by hematopoietic stem and progenitor cells (HSPCs). Expansion of PIGA and HLA class I mutant HSPCs have been linked to immune evasion from T-cell mediated pressures. We hypothesized that in analogy with antitumor immunity, the pathophysiological cascade of immune escape in IAA is initiated by immunoediting pressures and culminates with mechanisms of clonal evolution characterized by hits in immune recognition and response genes. To that end, we studied the genetic and transcriptomic make-up of the antigen presentation complexes in a large cohort of patients with IAA and paroxysmal nocturnal hemoglobinuria (PNH) by using single-cell RNA, high throughput DNA sequencing and single nucleotide polymorphism (SNP)-array platforms. At disease onset, HSPCs displayed activation of selected HLA class I and II-restricted mechanisms, without extensive inhibition of immune checkpoint apparatus. Using a newly implemented bioinformatic framework we found that not only class I but also class II genes were often impaired by acquisition of genetic aberrations. We also demonstrated the presence of novel somatic alterations in immune genes possibly contributing to the evasion from the autoimmune T-cells. In contrast, these hits were absent in myeloid neoplasia. These aberrations were not mutually exclusive with PNH and did not correlate with the accumulation of myeloid-driver hits. Our findings shed light on the mechanisms of immune activation and escape in IAA and define alternative modes of clonal hematopoiesis.

This study aimed to assess the prevalence of somatic mutations (SMs) in severe/very severe aplastic anemia (V/SAA) and transfusion-dependent nonsevere aplastic anemia (TD-NSAA) prior to immunosuppressive therapy (IST) and their impact on treatment efficacy. Next-generation sequencing was used to analyze 114 hematopoiesis-related genes at disease onset in 312 patients. SMs were detected in 17.9% of cases, involving 25 genes, most commonly DNMT3A (14, 20.9%) and BCOR (9, 13.4%). SMs were more frequent in patients over 40 years old, predominantly with a single mutation of low variant allele frequency (< 20%). Patients with SM were older and had lower lymphocyte counts. SMs did not significantly influence hematologic responses at 3, 6, or 12 months, relapse, progression, death, survival, or failure-free survival ( p  > 0.05). Grouping patients by mutated genes revealed no significant differences in IST efficacy, though Group I ( PIGA or BCOR/BCORL1 ) showed higher hematologic response rates in patients over 40 years of age. The cumulative incidence of clonal evolution was higher in Group II ( DNMT3A , TET2 , ASXL1 , FAT1 , or RUNX1 ), though not statistically significant. SMs in V/SAA and TD-NSAA were infrequent and did not affect IST outcomes or treatment decisions. However, the higher clonal evolution incidence in certain mutations warrants further research.

Clonal hematopoiesis (CH)—an expansion of blood cells derived from a single hematopoietic stem cell—is a defining feature of hematologic cancers, but recently CH was also found to be a frequent consequence of aging. When aging-associated CH results from acquisition of a somatic mutation in a driver gene associated with leukemia, and this mutation is present at a variant allele frequency of at least 0.02 (2%) yet the patient does not meet World Health Organization diagnostic criteria for a hematologic neoplasm, this state is termed clonal hematopoiesis of indeterminate potential (CHIP). CHIP is present in approximately 10% to 15% of people older than 70 years and more than 30% by age 85 years and represents a precursor state for neoplasia akin to monoclonal gammopathy of undetermined significance. Recently, CHIP was unexpectedly found to be an important risk factor for cardiovascular events, with accumulating evidence supporting a mechanism of accelerated atherogenesis as a result of vascular inflammation driven by clonally derived monocytes/macrophages. Risk factors for CHIP include aging, male sex, cigarette smoking, and a common germline variant in the telomere-associated gene TERT. Clonal hematopoiesis can also occur after cytotoxic chemotherapy or radiotherapy for a solid tumor, after hematopoietic stem cell transplant, in the context of aplastic anemia, or after induction chemotherapy for acute leukemia; in each setting, CH has distinct clinical implications. This review summarizes recent studies of CH and CHIP and outlines challenges in clinical management of affected patients.

Leukemias bearing heterozygous mutations in the SRSF2 splicing-factor-encoding gene can be therapeutically targeted by pharmacologic inhibition of residual spliceosome function. Mutations in genes encoding splicing factors (which we refer to as spliceosomal genes) are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) 1 , 2 , 3 . These mutations recurrently affect specific amino acid residues, leading to perturbed normal splice site and exon recognition 4 , 5 , 6 . Spliceosomal gene mutations are always heterozygous and rarely occur together with one another, suggesting that cells may tolerate only a partial deviation from normal splicing activity. To test this hypothesis, we engineered mice to express a mutated allele of serine/arginine-rich splicing factor 2 ( Srsf2 P95H )—which commonly occurs in individuals with MDS and AML—in an inducible, hemizygous manner in hematopoietic cells. These mice rapidly succumbed to fatal bone marrow failure, demonstrating that Srsf2 -mutated cells depend on the wild-type Srsf2 allele for survival. In the context of leukemia, treatment with the spliceosome inhibitor E7107 (refs. 7 , 8 ) resulted in substantial reductions in leukemic burden, specifically in isogenic mouse leukemias and patient-derived xenograft AMLs carrying spliceosomal mutations. Whereas E7107 treatment of mice resulted in widespread intron retention and cassette exon skipping in leukemic cells regardless of Srsf2 genotype, the magnitude of splicing inhibition following E7107 treatment was greater in Srsf2 -mutated than in Srsf2 -wild-type leukemia, consistent with the differential effect of E7107 on survival. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal gene mutations are preferentially susceptible to additional splicing perturbations in vivo as compared to leukemias without such mutations. Modulation of spliceosome function may thus provide a new therapeutic avenue in genetically defined subsets of individuals with MDS or AML.