APLASTIC ANEMIA

Aplastic anemia is an historic disease. The first patient was described by the young Paul Ehrlich in 1885, “anemia aplastique” originated with Vaquez in 1904, and its clinical features were described by Cabot and other pathologists in the early 20 th century. In the modern era, an almost uniformly fatal prognosis, mainly for young persons with sudden severe pancytopenia, has been reversed, with development of effective therapies for almost all patients. In the research laboratory understanding of pathophysiology has guided development of therapies. Marrow failure syndromes have been linked viral infection and environmental toxins, inherited and acquired genetic mutations, to the early events in leukemogenesis


Pathophysiologies
Three main pathophysiologies produce the pathology of an "empty" marrow (Figure 1).

Direct Marrow Damage.
Damage occurs most often iatrogenically, from chemotherapy and radiation.Marrow effects are dose-dependent and, at conventional doses, transient; other organ systems are affected; and spontaneous recovery is expected.Benzene, an inexpensive solvent, also damages hematopoiesis, and industrially exposed workers figured prominent in the early literature of aplastic anemia.Benzene now is a negligible risk factor, accounting for only a small etiologic fraction in most countries 1,2 .In China, rapidly industrialized and less regulated, benzene remains a workplace toxin 3,4 .Dosage is critical; workers with less intense and/or prolonged benzene exposure appear to suffer milder cytopenias, and they recover after

Immune aplastic anemia.
Almost all sporadic aplastic anemia, especially when severe and acute, appears to be immune-mediated.The strongest, most relevant evidence for an immune mechanism is the response of blood counts to a variety of immunosuppressive therapies and dependence of counts after recovery on maintenance calcineurin inhibitors 14,15 .Immune aplastic anemia lies in a spectrum of bone marrow and blood cell diseases (Figure 2A).Cytotoxic T cells have been the focus in studies of patient samples and in vitro.These cells appear functionally and phenotypically activated 16,17 , skewed to produce type 1 cytokines 18,19 , induce apoptosis via Fas/FasL 20 , and circulate as oligoclones. 21Acquired, somatic mutations in the STAT3 signaling pathway may be pathogenic in some aplastic anemia, as they are in large granular lymphocytosis, producing constitutively activated T cells 22 .Treg cells are decreased in patients with aplastic anemia and increase with hematologic response. 2324,25lastic anemia is associated with specific histocompatibility antigens 2627 .More striking is the presence of "escape clones", granulocytes with loss of the region of chromosome 6 that encompasses HLA alleles, in 10-15% of patients 2829,30 ; cells selected by absence of HLA, acquired by 6pLOH or somatic mutations, sustain hematopoiesis by clonal expansion 31 Immune escape has been hypothesized to explain clonal expansion of cells globally deficient in glycosylphosphoinositiol (GPI)-anchored proteins 32 in PNH, due to an acquired mutation in PIG-A in a stem cell.The GPI anchor itself has been suggested to be a target of the immune response. 33Autoantibodies, of uncertain significance, have been identified by high throughput screening of sera 3435 but the inciting antigen(s) for the dominant T cell response remain unknown.
Immune aplastic anemia can be modeled in mice: infusion of mis-matched donor lymphocytes leads to rapid hematopoietic failure and death. 3637Limited numbers of T cells specifically attack marrow cells, produce apoptosis through Fas/FasL engagement; type 1 cytokines have an active role in target cell death, directly for IFN-γ, indirectly for TNF-α; and Tregs are modulatory.

Hematopoiesis Stem Cell Number.
Aplastic anemia long has been regarded as the result of a profound deficit in hematopoietic stem and progenitor cells.The marrow is devoid of morphologic precursors to erythrocytes, granulocytes, and platelets.CD34 cells are almost completely absent in fixed biopsies or by flow cytometry.Colony forming cells for differentiated lineages and more immature multipotent cells are also extremely low in number.
None of the available measurements of hematopoiesis correlates closely with blood counts.More important, recovery of blood counts and of bone marrow function after immunosuppression and even more dramatically with growth factor stimulation indicates that stem cells are present even in the most deficient marrow.Assays to measure functional stem cells in humans are not quantitative, the contributions of true stem cells and more mature but still primitive multipotent progenitors to maintenance of hematopoiesis are controversial, and even changes with aging within the stem cell compartment have only been broadly defined.

Stem Cell Clonality.
Hematopoiesis in aplastic anemia is "clonal", but this is not a well-defined term 38 .Cancer is clonal: a tumor is derived from a single malignant cell.Clonality in marrow failure refers to the presence of populations originating from a single stem cell, which are easier to detect in circumstances of a failed bone marrow than in healthy individuals with hundreds of active stem cells.In aplastic anemia, benign clonal populations of granulocytes deficient in GPIanchored proteins or lacking HLA expression are frequent, presumably selected by survival under immune attack.Indeed, normal individuals have tiny numbers of leukocytes mutated in PIGA, and chromosomal clonal mosaicism is present in many normal tissues."Clonal evolution" in aplastic anemia is development of MDS or acute myeloid leukemia (AML), characterized by aneuploidy, usually loss of all or a portion of chromosome 7.That similar chromosome abnormalities feature in both acquired 39 and constitutional 40 aplastic anemia suggests that the marrow failure environment itself predisposes to their selection.
With next generation sequencing, clonality is apparent in leukocytes mutated in a specific gene, in most studies a "candidate" gene known to be recurrently mutated in MDS and AML. 41In aplastic anemia, such clonal populations are present in about 1/3 of patients, but in contrast to MDS and AML, a very limited set of genes (DNMT3A, ASXL1, and BCOR) is involved and the clone size (variant allele fraction) is small. 42,43The presence of a mutated clone in a patient associated with outcomes (BCOR and PIGA a favorable prognosis; mutations including DNMT3A and ASXL1, a worse prognosis). 42Paradoxically, mutated clones rarely appeared to drive evolution to a myeloid malignancy.

Telomeres.
Extremely short telomeres are typical of the patient with a genetic telomere disease.In immune aplastic anemia, telomere length may be decreased due to increased mitotic demand on a limited pool of stem cells. 44Telomere length at diagnosis has correlated with outcomes 45,46 , response to immunosuppression, 47 and evolution to MDS and AML. 45ccelerated telomere attrition precedes progression to monosomy 7. 48

Diagnosis
A fatty bone marrow remains basic to diagnosis, but sophisticated testing now can be directed at distinguishing among diverse pathophysiologies and discriminating among similar, sometimes overlapping diseases whi in the differential diagnosis (Fig. 2B).Accurate diagnosis is required for appropriate therapy and effective management.

Constitutional versus acquired bone marrow failure.
Genomic screening complements functional testing for Fanconi anemia (chromosomes after clastogenic stress) and telomeropathies (telomere length).However, comprehensive germline screening adds to the cost of the evaluation, results may not return to the clinician for several weeks, and a report can be difficult to interpret.Screening for the approximately 50 genes that cause constitutional marrow failure is particularly valuable in moderate and chronic pancytopenia, thrombocytopenia, and macrocytic anemia; absent a family history, physical stigmata, or evidence of organ involvement beyond the marrow, it is not likely to be positive in severe pancytopenia.Commercial testing reports "pathogenic" mutations, a determination that relies on continual reannotation of the literature and judgements based on amino acid changes and their location in conserved or functionally critical regions of a gene.Some base substitutions are infrequent polymorphisms in certain ethnic populations, and their significance is uncertain.Conversely, exome sequencing of candidate genes may not detect critical mutations in regulatory regions. 49,50Correlation of genomics with functional testing is desirable, but some telomeropathy patients have normal telomere length, short telomeres not below the first percentile can be difficult to interpret, and mosaicism due to reversion of a Fanconi anemia gene can lead to a normal chromosome study in peripheral blood.

Hypoplastic MDS versus aplastic anemia.
Acquired mutations are detected on genomic screens of recurrently mutated genes in MDS and AML.Such testing is valuable when MDS is suspected.Hypocellular MDS may be suggested from the bone marrow appearance, especially dyspoietic megakaryocytes, 51 and a normal or increased number of CD34 cells is not consistent with aplastic anemia.Flow cytometry enumerates CD34 cells and may show anomalous phenotypes indicating aberrant differentiation 52 .Genomics may be useful, as spliceosome gene mutations are prevalent in MDS but unusual in aplastic anemia, as is more than a single mutated gene. 53However, the genomic pattern of hypoplastic MDS, although distinct from normal or hypercellular MDS, is similar to the pattern in aplastic anemia, in the specific genes involved, the likelihood of only a single gene mutation, and smaller clone size. 54The finding of a DNMT3Aor ASXL1-mutated clone does not alter the diagnosis of aplastic anemia or likelihood of response to therapy.

PNH/aplastic anemia syndrome.
Screening for PNH is performed by flow cytometry, which precisely measures clone size as a proportion of GPI-anchored protein deficient cells by absence of specific antibody binding on erythrocytes and leukocytes.In hemolytic PNH, the clone is large, above 50% and sometimes approaching representation of all circulating cells from the mutated clone.A large clone also correlates with the risk of catastrophic clots and is an indication for anticomplement therapy with eculizumab; eculizumab resolves intravascular hemolysis and is effective as thrombosis prophylaxis.Clones are much smaller in aplastic anemia, requiring monitoring but not treatment; clinical PNH is unlikely to develop from tiny clones or without a clone at diagnosis 55

Treaments Bone marrow transplantation (BMT).
Replacement of a failed bone marrow is curative of the underlying disease.Transplant has been limited by its complications, graft rejection and graft-versus-host disease (GVHD), and the availability of suitable donors.
For immune aplastic anemia, transplant is always preferred in the young patient, and when undertaken expeditiously after diagnosis using a histocompatible sibling donor, results are excellent, with more than 90% long term survival in young children, 56,57 more than 80% in adolescents 58 , and a low rate of complications short-and long-term.While sibling donor transplant now is more frequent in older adults, results have not improved over several decades, remaining about 50% for recipients over 40 years of age 59 , almost 3-fold higher than in children. 60African-Americans also have poorer outcomes compared to Caucasians. 61Marrow is the preferred source due to more GVHD using peripheral blood. 62,63Rabbit ATG is often added to the conditioning regimen 64,65 , and radiation, especially in children, avoided.
7][68][69] In a comprehensive report of over 500 transplants, outcome measured as survival was not statistically inferior to conventional matched sibling transplants, but the frequency of serious GVHD was two-fold higher 67,68 Young age is a favorable factor for unrelated transplant as for sibling donor transplant.Children who have failed other therapies can receive unrelated grafts and have excellent survival, 95% in a multicenter British study. 70Outcomes are better with use of marrow rather than blood donor cells, ATG in the conditioning, younger donors, and a shorter interval from diagnosis. 67,68,71utcomes have been so good that some experts have advocated for first-line use of unrelated donors, 72,73 despite sometimes protracted delays in identifying and collecting donor cells.Late effects are more frequent in recipients after unrelated compared to sibling donor transplants. 74bilical cord transplantation also has been successful in aplastic anemia, mainly in children due to the relationship between donor inoculum cell numbers and recipient weight, with survival approximating 90%. 75,76Rates of GVHD are low; the major disadvantage of cord blood is delayed engraftment and prolonged neutropenia.Some protocols combine cord and mismatched bone marrow 77 A potential donor half matched to the patient should be present in virtually every family.As even single antigen disparities markedly affect outcomes of transplants, overcoming major histocompatibility differences had seemed an insuperable barrier.T cell depleting strategies, pre-transplant by cytotoxic drugs and biologics, and post-transplant with cyclophospamide 78 have been utilized to prevent GVHD.Extensive experience in Chinese centers and smaller series of patients transplanted in the United States and Europe shows excellent results (Table 2).Haploidentical transplant has been advocated in China as first treatment for children. 79; in Europe, with 1 year survival of about 77%, haploidentical transplant is recommended as second-line therapy. 80Long-term effects of the complicated regimens and mismatched immune system are unknown but possibly they will be ameliorated by the low prevalence and severity of graft-versus-host disease.
For the constitutional marrow failure syndromes, specific considerations relate to the underlying biology, in Fanconi anemia the sensitivity of cells in many organs to alkylating agents in conditioning regimens, and the natural history of these diseases, resulting in late cancers in Fanconi anemia and organ failure in telomere disease.Both the decision to transplant and timing of transplant can be difficult, due to slow progression of moderate hematopoietic failure and the uncertainty of highly variable disease outcomes without intervention. Worsening blood counts or evidence of progression to malignancy are obvious indications for transplant, and in some syndromes transplant has been remarkably effective. 84

Immunosuppression.
In the early years of transplant, occasional autologous recovery of patient marrow suggested that the antilymphocyte globulin employed in conditioning might have had a salutary effect.Combined with cyclosporine, anti-thymocyte globulin (ATG) leads to hematologic responses in about 2/3 of patients. 1480ATGs are complex, not fully defined mixtures of antibodies to human proteins.ATGs are relatively mildly lymphocyte depleting but subtle differences in mechanism of action appear important for efficacy.For example, rabbit ATG was much less efficacious than was horse ATG in a randomized controlled trial, in which a major difference in biologic effect was more severe depletion of CD4 cells and T regulatory cells following rabbit ATG. 25 Even with response, most patients do not recover normal blood counts.About 1/3 patients relapse or require cyclosporine long-term to maintain their response. 85; relapse usually responds to further immunosuppression. 86Responses and outcomes are better in children than in older adults. 87,8889Patients who do not respond to horse ATG may improve with second line rabbit ATG or alemtuzumab, a pan-T cell monoclonal antibody. 86mmunosuppressive therapy is less arduous than transplant and is available to all patients, but, in not replacing the affected marrow or immune system, late consequences of the disease can occur.Not infrequent relapse can be ameliorated but obligates the patient to long term cyclosporine.More serious is "clonal evolution", the later development of MDS or AML, even after stable blood count recovery.Clonal evolution most often manifests as a cytogenetic abnormality, usually loss of all or part of chromosome 7 39 and occurs in about 15% of aplastic anemia patients over the decade following initial immunosuppression. 14hromosome 7 aneuploidy has a poor prognosis and triggers efforts to transplant.

Stem Cell Stimulation.
Attempts to improve on ATG by addition of androgens, granulocyte colony stimulating factor, mycophenolate, or rapamycin have not altered response rates or long-term outcomes.Hematopoietic growth factors are ineffective in aplastic anemia.It was therefore unexpected when eltrombopag, a synthetic mimetic of thrombopoietin, showed activity in patients with refractory aplastic anemia, about half of whom responded with robust trilineage improvements in blood counts, most durable after discontinuation of drug. 9091Eltrombopag has been relabeled for this indication.When eltrombopag was added to initial standard immunosuppression, it markedly increased the overall response rate to about 80% and the complete response rate to about 50%, with patients often showing more rapid than expected hematologic recovery. 46To date, the rates of relapse and evolution to myeloid malignancies appear similar or lower than in historical controls treated with immunosuppression alone.
Increased bone marrow cellularity, CD34 cell and progenitor numbers suggest a direct effect of eltrombopag on marrow stem cells. 46Thrombopoietin concentrations in the blood of aplastic anemia patients are very high 92,93 , but eltrombopag may evade a block to receptor engagement in the presence of interferon-γ (Alvarado and Larochelle, personal communication).

Androgens.
Androgens are historic therapy for marrow failure syndromes.While generally regarded as much less efficacious in severe aplastic anemia than are immunosuppressive strategies, androgens are standard care for many constitutional syndromes.Sex hormones increase expression of the gene for telomerase in cell culture 94 and in mice. 95In a recent prospective trial, high doses of danazol, a synthetic androgen, improved blood counts in patients with telomere disease and also appeared to reverse accelerated telomere attrition. 96ovisional treatment algorithms are provided in Figure 3.

Conclusion
Aplastic anemia is a remarkable story of success in the clinic and the laboratory, with implications beyond bone marrow failure.Its etiologies relate to common environmental toxins, to specific viral infections, and to genes affecting basic cellular mechanisms; the role of the immune system has been appreciated as both potent and subtle.Most gratifying, treatments for the patient with immune aplastic anemia have improved remarkably over the last several decades, due to the development of better transplant and immunosuppression Pathophysiologies of aplastic anemia.The common pathology of the bone marrow replaced by fat can result from chemical or physical damage (iatrogenic; benzene); immune destruction (mainly T cells); and as a constitutional defect in genes important in maintenance of cell integrity and immune regulation.HGF=hematopoietic growth factors; BMT= bone marrow transplantation; IST=immunosuppression.

Figure 2 .
Figure 2.Aplastic anemia in relationship to other diseases.A) Venn diagram emphasizing overlap of aplastic anemia, both diagnostic and pathophysiologic, with PNH, MDS, and constitutional marrow failure syndromes, as well as to other immune-mediated diseases in which a single

Figure 3 .
Figure 3.Treatment algorithms for A) children and B) adults with immune aplastic anemia.Treatment strategies are not fixed, due to developing results, especially long-term, with the use of new agents like eltrombopag and transplants from alternative donors; proposed approaches are indicated with dashed arrows.The ordinate bars represent the stages of treatment: days to diagnose and stabilize the severely pancytopenic patient; months to select, implement, and complete definitive therapy; and years of monitoring for responses and complications.The ability to salvage the extremely susceptible neutropenic patient105 is fundamental to longterm outcomes.Undesirable delays from diagnosis to transplant should be avoidable when outcomes from immunosuppression are clear in 3-6 months.Patients who fail immunosuppression70,106103  or who develop complications 107 can do well with second-line transplant.IST=immunosuppression, BMT= bone marrow transplantation, MUD=matched unrelated donor transplant.

Table 1 .
Constitutional Marrow Failure Syndromes Presenting in Adults N Engl J Med. Author manuscript; available in PMC 2019 April 16.

Table 2A .
Hematopoietic Stem Cell Transplant in Severe Aplastic Anemia: Matched Family and Matched Unrelated Donors aGVHD=acute graft-versus-host disease, cGVHD=chronic GVHD, gr=grade, IBMTR=International Bone Marrow Transplant Registry, EGBMT=European Group for Bone Marrow Transplant, MFD=matched family donor, MUD=matched unrelated donor, RCT=randomized control trial, Cy=cyclophosphamide, CsA=cyclosporine, MTX=methotrexate, FLU=fludarabine, * Randomized control trial (RCT) comparing Cy and Cy + ATG, between which there were no statistically significant differences.For simplicity, only CTX+ATG arm data are shown as this is more common conditioning regimen.Only data from alemtuzumab-treated patients are shown; MSD and MUD are combined, as they were in the original.
** N Engl J Med. Author manuscript; available in PMC 2019 April 16.
N Engl J Med. Author manuscript; available in PMC 2019 April 16.