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Clonal evolution to secondary paroxysmal nocturnal hemoglobinuria (PNH) or myeloid neoplasia (MN) represents one of the long-term complications of patients with aplastic anemia (AA). The recent evidence in the field of immunology and the application of next-generation sequencing have shed light on the molecular underpinnings of these clonal complications, revealing clinical and molecular risk factors as well as potential immunological players. Particularly, whether MN evolution represents a failed tumor surveillance or a maladaptive recovery is still a matter of controversy in the field of bone marrow failure syndromes. However, recent studies have explored the precise dynamics of the immune-molecular forces governing such processes over time, generating knowledge useful for potential early therapeutic strategies. In this review, we will discuss the immune pathophysiology of AA and the emergence of clonal hematopoiesis with regard to the adaptive and maladaptive mechanisms at the basis of secondary evolution trajectories operating under the immune pressure. [ABSTRACT FROM AUTHOR]

Background Aplastic anaemia is a bone-marrow-failure syndrome characterised by low blood-cell counts and fatty bone marrow. In most cases, no obvious aetiological factor can be identified. However, clinical responses to immunosuppression strongly suggest an immune pathophysiology. Methods To test the hypothesis that aplastic anaemia results from antigen-specific lymphocyte attack against haemopoietic tissue, we analysed effector immunity, seeking especially dominant specific T-cell responses. Blood samples from 54 patients with aplastic anaemia were subjected to flow cytometry to define T-cell-receptor V -chain usage and expansion of particular V subsets. We measured the size distribution of the complementarity determining region 3 (CDR3) for expanded V subsets, then cloned and sequenced skewed, oligoclonal, or monoclonal peaks. Findings Expanded V subsets were identified in almost all the patients. Over-represented V subsets from CD8-positive cells showed oligoclonal or monoclonal CDR3 size patterns. The CDR3 sequence repertoire in aplastic anaemia showed much redundancy compared with healthy donors. We identified patient-specific putative pathogenetic clonotypes that were not detectable in controls. In selected patients who were assessed longitudinally, these clonotypes were quantitatively related to disease activity. Selective killing of autologous haemopoietic progenitors by the V -specific lymphocyte population was shown in one patient. These apparently pathogenetic CDR3 sequences showed homology between individuals, suggesting a role for a "semi-public" immune response in the pathophysiology of aplastic anaemia. Interpretation In-vivo dominant clonal immune response can be identified in many patients with aplastic anaemia, which is evidence for an underlying antigen-driven immune process. [ABSTRACT FROM AUTHOR]

Prior to the introduction of effective therapies, the high mortality rates of severe aplastic anemia (AA) precluded recognition of late complications of this disease. Once the survival of AA improved, observation of clonal evolution raised questions as to whether the development of secondary myelodysplastic syndrome (MDS) is a part of the extended natural history of the disease or is related to the therapies applied. Clinical features of myelodysplasia and AA can overlap, and typical MDS may evolve as a complication of AA. Common pathophysiologic elements operate in these diseases and are subject to many studies and theories as to what mechanisms in AA may lead to the late evolution of MDS. Similarly, AA has been hypothesized to be a reflection of an over-reactive immune response triggered by the appearance of genetically altered and/or phenotypically abnormal dysplastic clones. Hypocellular variants of myelodysplasia and responsiveness of certain forms of MDS to immunosuppressive regimens serve as the most appealing examples of the intricate and close pathophysiologic relationship of this disease with AA. The diagnosis of clonal evolution in the course of AA can be obvious if secondary cytopenia involves hypercellularity and a high percentage of blasts. In addition, the occurrence of a new karyotypic defect objectively heralds the progression of disease to MDS. However, the diagnostic imprecision of dysplasia recognition in the context of marrow hypocellularity, inability to obtain informative cytogenetics, and a high proportion of MDS cases with normal karyoptype have hampered studies designed to determine the frequency and timing of MDS evolution in AA. In addition, the diagnostic criteria and definitions used are not unified. While some centers recognize that the abnormal karyotype does not preclude the diagnosis of AA; in others, the diagnosis of AA includes the presence of normal karyoptype. Many typical features of dysplastic evolution in AA have been clarified. For example, karyotypes most frequently encountered in MDS secondary to AA involve chromosomes 6, 7 and 8. The evolution rates seem to be in the range of 10 - 15% in 10 years, but there are no predictive clues as to which patients are at greatest risk for this complication. Study of the mechanisms of clonal evolution in AA may help understand the pathophysiology of other forms of MDS and leukemia and also the mechanisms of antileukemic surveillance. Clinically, identification of patients at increased risk for clonal complications may influence the choice of therapies applied. [ABSTRACT FROM AUTHOR]

Profound cytopenia involving all blood lineages, a hallmark of aplastic anemia (AA), can result in devastating morbidity and high mortality. Although various etiologies and distinct pathophysiologic mechanisms may be involved, a profound defect in the stem cell compartment is a unifying feature in most patients with AA. As a stem cell disease, AA is very instructive and provides insights into the function and quantity of normal hematopoietic stem cells and their ability to regenerate. Pathophysiologically, understanding of AA may reveal mechanisms as to the evolution of other related bone marrow failure syndromes such as paroxysmal nocturnal hemoglobinuria and myelodysplasia—clonal diseases of hematopoiesis associated with defective stem cells. Conversely, constitutional forms of AA occurring in association with Fanconi anemia and dyskeratosis congenita demonstrate the role of specific genes and pathways in the dysfunction of the stem cells leading to the failure of the stem cell compartment.The acquired mechanisms resulting in depletion of stem cells in AA may involve fundamental pathways such as apoptosis and senescence as well as exhaustion of proliferative capacity or excessive differentiation. Inherent in the paucity of the bone marrow in AA, the study of the stem cells in AA has been very difficult due to their natural rarity and disease-specific contraction of the stem cell pool. Despite these scientific challenges, laboratory studies and systematic clinical observation provide valuable information of significance beyond its specific application to AA. [Copyright &y& Elsevier]

Introduction: Complement C5 inhibitor eculizumab is the first approved treatment for paroxysmal nocturnal hemoglobinuria (PNH), a rare hematologic disorder caused by uncontrolled terminal complement activation. Approximately 50% of patients with aplastic anemia (AA) have PNH cells. Limited data are available for patients with AA-PNH taking concomitant immunosuppressive therapy (IST) and eculizumab. Methods: Data from the International PNH Registry (NCT01374360) were used to evaluate the safety and effectiveness of eculizumab and IST in patients taking IST followed by concomitant eculizumab (IST + c-Ecu) or eculizumab followed by concomitant IST (Ecu + c-IST). Results: As of January 1, 2018, 181 Registry-enrolled patients were included in the eculizumab effectiveness analyses (n = 138, IST + c-Ecu; n = 43, Ecu + c-IST); 87 additional patients received IST alone. Reductions from baseline with eculizumab were observed in the least squares mean lactate dehydrogenase ratio (IST + c-Ecu, −3.4; Ecu + c-IST, −3.5); thrombotic event incidence rates were similar between groups (IST + c-Ecu, 1.3; Ecu + c-IST, 0.7). Red blood cell transfusion rate ratios decreased from baseline for IST + c-Ecu (0.7) and increased for Ecu + c-IST (1.2); there were none for IST alone. Hematological parameters generally improved for IST + c-Ecu and IST alone, and changed minimally or worsened for Ecu + c-IST. Safety signals were generally consistent with those previously described for the respective therapies. Discussion/conclusion: Although some intergroup differences were seen, concomitant eculizumab and IST were safe and effective regardless of treatment sequence. [ABSTRACT FROM AUTHOR]

Abstract: Acquired amegakaryocytic thrombocytopenia and pure red cell aplasia rarely occur concurrently. We report a case in which these disorders were associated with an occult large granular lymphocyte leukemia. The peripheral blood cytopenias improved after glucocorticoids and intravenous immunoglobulin were administered, and response was maintained with cyclosporine. Large granular lymphocyte leukemia should be suspected in the setting of unexplained bone marrow failure. [Copyright &y& Elsevier]

The basis for the vast recognition spectrum of the T-cell receptor (TCR) can be determined by the rearrangement and recombination of the variable, diversity and joining regions of the variable portions of β (B) and α (A) chains as well as their recombination and modification. Analysis of the TCR rearrangement has been routinely used to detect clonality for the diagnosis of lymphoid malignancies. However, molecular analysis of the TCR repertoire can be a powerful tool in the study of T-cell responses to pathogens and in autoimmune diseases. The concept of the oligoclonality in the context of cellular immune responses is based on the presence of immunodominant T-cell clones within distinct T-cell subpopulations used for analysis. Under normal circumstances, a limited number of clones undergo periodic expansions in reaction to foreign antigens. Under pathologic conditions, though, the derailment of immune regulation allows expansions of specific and potentially pathogenic T-cell clones. For example, large granular lymphocyte (LGL) leukemia illustrates an extreme expansion of a single T-cell clone associated with a distinct autoimmune pathology, which suggests an exaggerated clonal response to a specific antigenic target. In immune-mediated bone marrow failure syndromes, clonal rearrangement of the TCR cannot be detected in unseparated blood or marrow. Nevertheless, individual T-cell clones can significantly expand and may allow for demonstration of oligoclonality in selected T-cell populations. These subpopulations are defined, for example, by a specific β (B)-chain usage or other phenotypic markers. Given the diversity of the TCR recognition spectrum, the task of identifying immunodominant clonotypes derived from unique complementarity determining region-3 (CDR3) sequences is very complex. However, expanded T-cell clones likely represent immunodominant responses which can be detected on the molecular level using analysis of the individual TCR VB-chain representation, CDR3 size fragment skewing, and determination of the frequency of individual clonotypic sequences. In the future, TCR VB clonotypes may be applied as a diagnostic tool, analogous to serologic markers. As an investigative tool in hematology, molecular analysis of the TCR utilization pattern and the detection of immunodominant clonotypes represents a novel approach in the study of immune-mediated hematologic diseases, such as aplastic anemia (AA), some forms of myelodysplasia (MDS), anti-leukemic immune surveillance, graft-versus-leukemia effects and graft-versus-host disease (GvHD). [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the effects of eculizumab on transfusions and thrombotic events (TEs) in patients with and without prior history of transfusion in the International Paroxysmal Nocturnal Hemoglobinuria (PNH) Registry. Methods: Registry patients enrolled on or before January 1, 2018, initiated on eculizumab no more than 12 months prior to enrollment, having known transfusion status for the 12 months before eculizumab initiation, and ≥12 months of Registry follow‐up after eculizumab initiation, were included. Results: Eculizumab treatment was associated with a 50% reduction in transfusions in patients with a transfusion history (10.6 units/patient‐year before eculizumab vs 5.4 after; P <.0001), with greater reduction observed in those with no history of bone marrow disease vs those with bone marrow disease. Mean lactate dehydrogenase levels decreased from a mean of 6.7 to 1.4 times the upper limit of normal (ULN) in patients with transfusion history and from 5.1 to 1.2 times ULN in those with no transfusion history. TE and major adverse vascular event rates also decreased by 70% in patients with and without history of transfusion. Conclusions: The benefit of eculizumab therapy does not appear to be limited to any group defined by transfusion history or bone marrow disease history. [ABSTRACT FROM AUTHOR]

The article focuses on the study of aplastic anaemia (AA), pure red cell aplasia (PRCA), pure white cell aplasia (PWCA) and immune cytopenias (IC) related to T-cell large-granular lymphocytic leukaemia (T-LGL) with the aspects of immune-pathogenesis. It mentions that polyclonal, clonally skewed or clonal cytotoxic T-lymphocytes (CTLs) can mediate cytopenias.

Abstract: Large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of antigen-activated cytotoxic T cells (CTL). Patients frequently exhibit seroreactivity against a human T-cell leukemia virus (HTLV) epitope, BA21. Aplastic anemia, paroxysmal nocturnal hemoglobinuria and myelodysplastic syndrome are bone marrow failure diseases that can also be associated with similar aberrant CTL activation (LGL-BMF). We identified a BA21 peptide that was specifically reactive with LGL leukemia sera and found significantly elevated antibody reactivity against the same peptide in LGL-BMF sera. This finding of shared seroreactivity in LGL-BMF conditions and LGL leukemia suggests that these diseases might share a common pathogenesis. [Copyright &y& Elsevier]

In aplastic anemia (AA), contraction of the stem cell pool may result in oligoclonality, while in myelodysplastic syndromes (MDS) a single hematopoietic clone often characterized by chromosomal aberrations expands and outcompetes normal stem cells. We analyzed patients with AA (N = 93) and hypocellular MDS (hMDS, N = 24) using single nucleotide polymorphism arrays (SNP-A) complementing routine cytogenetics. We hypothesized that clinically important cryptic clonal aberrations may exist in some patients with BM failure. Combined metaphase and SNP-A karyotyping improved detection of chromosomal lesions: 19% and 54% of AA and hMDS cases harbored clonal abnormalities including copy-neutral loss of heterozygosity (UPD, 7%). Remarkably, lesions involving the HLA locus suggestive of clonal immune escape were found in 3 of 93 patients with AA. In hMDS, additional clonal lesions were detected in 5 (36%) of 14 patients with normal/noninformative routine cytogenetics. In a subset of AA patients studied at presentation, persistent chromosomal genomic lesions were found in 10 of 33, suggesting that the initial diagnosis may have been hMDS. Similarly, using SNP-A, earlier clonal evolution was found in 4 of 7 AA patients followed serially. In sum, our results indicate that SNP-A identify cryptic clonal genomic aberrations in AA and hMDS leading to improved distinction of these disease entities. [ABSTRACT FROM AUTHOR]

Although recent advances in therapy offer the promise for improving survival in patients with severe aplastic anemia (SAA), the small size of the patient population, lack of a mechanism in North America for longitudinal follow-up of patients, and inadequate cooperation among hematologists, scientists, and transplant physicians remain obstacles to conducting large studies that would advance the field. To address this issue, the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) convened a group of international experts in March 2010 to define the most important questions in the basic science, immunosuppressive therapy (IST), and bone marrow transplantation (BMT) of SAA and propose initiatives to facilitate clinical and biologic research. Key conclusions of the working group were: (1) new patients should obtain accurate, expert diagnosis and early identification of biologic risk; (2) a population-based SAA outcomes registry should be established in North America to collect data on patients longitudinally from diagnosis through and after treatment; (3) a repository of biologic samples linked to the clinical data in the outcomes registry should be developed; (4) innovative approaches to unrelated donor BMT that decrease graft-versus-host disease are needed; and (5) alternative donor transplantation approaches for patients lacking HLA-matched unrelated donors must be improved. A partnership of BMT, IST, and basic science researchers will develop initiatives and partner with advocacy and funding organizations to address these challenges. Collaboration with similar study groups in Europe and Asia will be pursued. [Copyright &y& Elsevier]

Abstract: While a majority of patients with refractory anemia with ring sideroblasts and thrombocytosis harbor JAK2V617F and rarely MPLW515L, JAK2/MPL-negative cases constitute a diagnostic problem. 23 RARS-T cases were investigated applying immunohistochemical phospho-STAT5, sequencing and SNP-A-based karyotyping. Based on the association of TET2/ASXL1 mutations with MDS/MPN we studied molecular pattern of these genes. Two patients harbored ASXL1 and another 2 TET2 mutations. Phospho-STAT5 activation was present in one mutated TET2 and ASXL1 case. JAK2V617F/MPLW515L mutations were absent in TET2/ASXL1 mutants, indicating that similar clinical phenotype can be produced by various MPN-associated mutations and that additional unifying lesions may be present in RARS-T. [Copyright &y& Elsevier]

The efficacy of immunosuppressive therapy in aplastic anemia (AA) provides the strongest argument to support its immune-mediated pathophysiology. While several immunosuppressive effects of antithymocyte globulin (ATG) can be demonstrated in vitro, some reports have implied that the activity of ATG in AA may be rather due to a variety of positive hematopoietic effects. We studied the effects of horse (h) and rabbit (r) ATG on marrow progenitors in vitro. Both types of ATG bound to CD34 cells and, in colony assays performed with total marrow cells, hATG had a dose-dependent, triphasic effect, with maximal increase in colony formation between 1 and 10?µg/ml and inhibition between 100 and 1000?µg/ml. As determined using CD34 cells, these effects did not require accessory cells. rATG showed similar activity, but was about 10-fold more potent than hATG. In the presence of complement, no increased cytotoxicity was observed. At concentrations equivalent to those measured in patients immediately after infusion, ATG showed moderate suppression of colony formation, while the stimulatory concentrations in vitro correspond to those seen in vivo within the first weeks after ATG administration. In control experiments, the patterns of the biologic effects of preimmune rIgG or hIgG preparations were similar to those of rATG and hATG, indicating a nonspecific nature of the effects of ATG on progenitor cells. Biological activity in methylcellulose cultures was observed with the F(ab)2 fragments but was not found in purified Fc IgG. In summary, the spectrum of effects of ATG on hematopoietic progenitors is dependent upon the concentrations of ATG and may not be related to its antigenic specificity.The Hematology Journal (2004) 5, 255-261. doi:10.1038/sj.thj.6200398 [ABSTRACT FROM AUTHOR]