Chapter 35: Aplastic Anemia: Acquired and Inherited

DEFINITION AND HISTORY

Aplastic anemia is a clinical syndrome that results from a marked diminution of marrow blood cell production. The decrease in hematopoiesis results in reticulocytopenia, anemia, granulocytopenia, monocytopenia, and thrombocytopenia. The diagnosis usually requires the presence of pancytopenia with a neutrophil count fewer than 1500/μL (1.5 × 10⁹/L), a platelet count fewer than 50,000/μL (50 × 10⁹/L), a hemoglobin concentration less than 10 g/dL (100 g/L), and an absolute reticulocyte count fewer than 40,000/μL (40 × 10⁹/L), accompanied by a hypocellular marrow without abnormal or malignant cells or fibrosis.¹ For the purpose of therapeutic decision making, comparative clinical trials, and international sharing of data, the disease has been stratified into moderately severe, severe, and very severe acquired aplastic anemia based on the blood counts (especially the neutrophil count) and the degree of marrow hypocellularity (Table 35–1). Most cases of aplastic anemia are acquired; fewer cases are the result of an inherited disorder, such as Fanconi anemia, Shwachman-Diamond syndrome, and others (see “Hereditary Aplastic Anemia” below).

Aplastic anemia was first recognized by Paul Ehrlich in 1888.² He described a young, pregnant woman who died of severe anemia and neutropenia. Thrombocytopenia was difficult to measure and the role of blood dust (platelets) was controversial at that time. Autopsy examination revealed a fatty marrow with essentially no hematopoiesis. The name aplastic anemia was subsequently applied to this disease by Chauffard, a French hematologist, in 1904,³ and although an anachronistic term because the morbidity is the result of pancytopenia, especially neutropenia and thrombocytopenia, the designation is entrenched in medical usage. For the next 40 years, many conditions that caused pancytopenia were confused with aplastic anemia based on incomplete or inadequate histologic study of the patient’s marrow.⁴ The development of improved instruments for percutaneous marrow biopsy in the last half of the 20th century improved diagnostic precision. In 1972, Thomas and his colleagues established that marrow transplantation from a histocompatible sibling donor could cure the disease.⁵ The disease initially was thought to result from an atrophy or chemical injury of primitive marrow hematopoietic cells. The unexpected recovery of marrow recipients who were given immunosuppressive conditioning therapy but who did not engraft with donor stem cells raised the possibility that the disease may not be intrinsic to primitive hematopoietic cells but the result of a suppression of hematopoietic cells by immune cells, notably T lymphocytes.⁶ The requirement to treat the recipient of a marrow transplant from an identical twin with immunosuppressive conditioning therapy for optimal results of transplant, buttressed this concept.⁷ This supposition was confirmed by a clinical trial that established antilymphocyte globulin (ALG) capable of ameliorating the disease in the majority of patients.⁸ Since that time, compelling evidence for a cellular autoimmune mechanism has accumulated (see “Etiology and Pathogenesis” below).

EPIDEMIOLOGY

The International Aplastic Anemia and Agranulocytosis Study and a French study found the incidence of acquired aplastic anemia to be approximately 2 per 1,000,000 persons per year.^1,9 This annual incidence has been confirmed in studies in Spain (Barcelona),¹⁰ Brazil (State of Parana),¹¹ and Canada (British Columbia).¹² The highest frequency of aplastic anemia occurs in persons between the ages of 15 and 25 years; a second peak occurs between the ages of 65 and 69.¹ Aplastic anemia is more prevalent in the Far East where the incidence is approximately 7 per 1,000,000 in parts of China,¹³ approximately 4 per 1,000,000 in sections of Thailand,¹⁴ approximately 5 per 1,000,000 in areas of Malaysia,¹⁵ and approximately 7 per 1,000,000 among children of Asian descent living in Canada.¹² The explanation for a twofold or greater incidence in the Orient compared to the Occident may be multifactorial,¹⁶ but a predisposition gene or genes is a likely component.^12,17 Studies have not established the use of chloramphenicol in Asia as a cause. Poorly regulated exposure of workers to benzene is a factor,¹⁸ but the attributable risk from benzene and other toxic exposures does not explain the magnitude of the difference in the incidence in Asia compared to that in Europe and South America.^16,17 A relationship to impure water use in Thailand has led to speculation of an infectious etiology, although no agent, including seronegative hepatitis, a known association with the onset of acquired aplastic anemia,¹⁶ has been identified. Seronegative viral hepatitis is a forerunner of approximately 7 percent of cases of acquired aplastic anemia.^17,19 The male-to-female incidence ratio of aplastic anemia in most studies is approximately one.¹⁷

ETIOLOGY AND PATHOGENESIS

Table 35–2 lists the conditions associated with aplastic anemia.

The final common pathway to the clinical disease is a decrease in blood cell formation in the marrow. The number of marrow CD34+ cells (multipotential hematopoietic progenitors) and their derivative colony-forming unit–granulocyte-macrophage (CFU-GM) and burst-forming unit–erythroid (BFU-E) are reduced markedly in patients with aplastic anemia.^20-23 Long-term culture-initiating cells, an in vitro surrogate assay for hematopoietic stem cells, also are reduced to approximately 1 percent of normal values.²³ Potential mechanisms responsible for acquired marrow cell failure include (1) cellular or humoral immune suppression of the marrow multipotential cells, (2) progressive erosion of chromosome telomeres, (3) direct toxicity to hematopoietic multipotential or stem cells, (4) a defect in the stromal microenvironment of the marrow required for hematopoietic cell development, and (5) impaired production or release of essential multilineage hematopoietic growth factors. There is little experimental evidence for a stromal microenvironmental defect or a deficit of critical hematopoietic growth factors or their receptors. Telomerase mutations with consequent telomere shortening may be involved in as many as 40 percent of patients.²⁴ A susceptibility to the development of aplastic anemia is present in persons with certain human leukocyte antigen (HLA) types, such as HLA-DR15.²⁴

Deficiencies in telomere repair could predispose to aplastic anemia by affecting the size of the multipotential hematopoietic cell compartment and by decreasing the multipotential cell’s response to marrow injury, and could play a role in the evolution of aplastic anemia to a clonal myeloid disease by contributing to genomic instability.²⁵ Reduced hematopoiesis in most cases of aplastic anemia results from cytotoxic T-cell–mediated immune suppression of very early CD34+ hematopoietic multipotential progenitor or stem cells.²⁶ A small fraction of cases is initiated by a toxic exposure, drug exposure, or viral infection, and in these cases the pathogenesis also may relate to autoimmunity as there is evidence of immune dysfunction in seronegative hepatitis, after benzene exposure, and many such patients respond to anti–T-cell therapy.²⁶

Autoreactive Cytotoxic T Lymphocytes

In vitro and clinical observations have resulted in the identification of a cytotoxic T-cell–mediated attack on multipotential hematopoietic cells in the CD34+ cellular compartment as the basis for most cases of acquired aplastic anemia.²⁷ Cellular immune injury to the marrow after drug-, viral-, or toxin-initiated marrow aplasia could result from the induction of neoantigens that provoke a secondary T-cell-mediated attack on hematopoietic cells. This mechanism could explain the response to immunosuppressive treatment in cases that follow exposure to an exogenous agent. Spontaneous or mitogen-induced increases in mononuclear cell production of interferon-γ,^28,29 interleukin (IL)-2,²⁹ and tumor necrosis factor-α (TNF-α)^30,31 occur. These factors are inhibitory to hematopoietic cell development. Elevated serum levels of interferon-γ are present in 30 percent of patients with aplastic anemia, and interferon-γ expression has been detected in the marrow of most patients with acquired aplastic anemia.³² Addition of antibodies to interferon-γ enhances in vitro colony growth of marrow cells from affected patients.³³ Long-term marrow cultures manipulated to elaborate exaggerated amounts of interferon-γ, markedly reduced the frequency of long-term culture-initiating cells.²⁶ These observations indicate that acquired aplastic anemia is the result of cellular immune-induced apoptosis of primitive CD34+ multipotential hematopoietic progenitors, mediated by cytotoxic T lymphocytes, in part, through the expression of T-helper type 1 (Th1) inhibitory cytokines, interferon-γ, and TNF-α (Fig. 35-1).³⁴ The secretion of interferon-γ is a result of the upregulation of the regulatory transcription factor T-bet,³⁵ and apoptosis of CD34+ cells is, in part, mediated through a FAS-dependent pathway.²⁶ Because HLA-DR2 is more prevalent in patients with aplastic anemia, antigen recognition may be a factor in those patients. A variety of other potential factors have been found in some patients, including nucleotide polymorphisms in cytokine genes, overexpression of perforin in marrow cells, and decreased expression of SLAM-associated protein (SAP), a modulator protein that inhibits interferon-γ secretion.²⁶

A decrease in regulatory T cells (CD4+CD25+FoxP3+) contributes to the expansion of an autoreactive CD8+CD28− T-cell population, which induces apoptosis of autologous hematopoietic multipotential hematopoietic cells.^36-38 T-regulatory cells are a component of the immune system that suppress immune responses of other cells. They provide a “stop” for immune reactions that have achieved their purpose. They also play a role in preventing autoimmune reactions (Chap. 76). One mouse model of immune-related marrow failure, induced by infusion of parental lymph node cells into F1 hybrid recipients, caused a fatal aplastic anemia. The aplasia could be prevented by immunotherapy or with monoclonal antibodies to interferon-γ and TNF-α.²⁶ Another mouse model of aplastic anemia induced by the infusion of lymph node cells histoincompatible for the minor H antigen, H60, resulted from the expansion of H60-specific CD8 T cells in recipient mice. The result was severe marrow aplasia. The effect of the CD8 T cells could be abrogated by either immunosuppressive agents or administration of CD4+CD25+ regulatory T cells,³⁹ providing additional experimental evidence for the role of regulatory T cells in the prevention of aplastic anemia. There are two subsets of regulatory T cells, Treg A and Treg B, with distinct phenotypes.⁴⁰ Treg B cells have a greater expression of CD95, CCR4 and CD45RO antigens than do Treg A cells. Aplastic anemia patients responding to immunotherapy have a predominance of Treg B cells.

Several putative target antigens on affected hematopoietic cells have been identified. Autoantibodies to one putative antigen, kinectin, have been found in patients with aplastic anemia. T cells, responsive to kinectin-derived peptides, suppress granulocyte-monocyte colony growth in vitro. However, in these studies cytotoxic T lymphocytes with that specificity were not isolated from patients.⁴¹ Thus, the putative antigen(s) that is the target of the autoreactive T cells has not been identified.

Telomere Shortening

A relationship between acquired aplastic anemia and hereditary aplastic anemia (Fanconi anemia or dyskeratosis congenita) in some patients has been suggested because the defects in telomerase and telomere repair, characteristic of Fanconi anemia and dyskeratosis congenita are shared in some adult patients with aplastic anemia, but in these cases there is no family history of such a disorder and no phenotypic abnormalities that characterize the hereditary disorders (see “Fanconi Anemia” and “Dyskeratosis Congenita” below). Telomeres shorten physiologically with age as telomerase becomes less active. T-cell–mediated acquired aplastic anemia is associated with telomere shortening which could reflect an inherited defect in telomerase or a senescent erosion of activity. The telomerase mechanism consists of a telomerase reverse transcriptase (TERT); an RNA template for TERT, the telomerase RNA component (TERC), and other stabilizing proteins.^42,43 Cells with shortened telomeres normally undergo apoptosis unless DNA repair mechanisms are impaired allowing the development of aneuploidy and neoplastic transformation.

Drugs

Chloramphenicol is the most notorious drug documented to cause aplastic anemia. Although this drug is directly myelosuppressive at very high dose because of its effect on mitochondrial DNA, the occurrence of aplastic anemia appears to be idiosyncratic, perhaps related to an inherited sensitivity to the nitroso-containing toxic intermediates.⁴⁴ This sensitivity may produce immunologic marrow suppression, as a substantial proportion of affected patients respond to treatment with immunosuppressive therapy.⁴⁵ The risk of developing aplastic anemia in patients treated with chloramphenicol is approximately 1 in 20,000, or 25 times that of the general population.⁴⁶ Although its use as an antibiotic has been largely abandoned in industrialized countries, global reports of fatal aplastic anemia continue to appear with topical or systemic use of the drug.

Epidemiologic evidence established that quinacrine (Atabrine) increased the risk of aplastic anemia.⁴⁷ This drug was administered to all U.S. troops in the South Pacific and Asiatic theaters of operations as prophylaxis for malaria during 1943 and 1944. The incidence of aplastic anemia was 7 to 28 cases per 1,000,000 personnel per year in the prophylaxis zones, whereas untreated soldiers had 1 to 2 cases per 1,000,000 personnel per year. The aplasia occurred during administration of the offending agent and was preceded by a characteristic rash in nearly half the cases. Many other drugs have been reported to increase the risk of aplastic anemia, but owing to incomplete reporting of information and the infrequency of the association, the spectrum of drug-induced aplastic anemia may not be fully appreciated. Table 35–3 is a partial list of drugs that have been implicated.^48-56

Many of these drugs are known to also induce selective cytopenias, such as agranulocytosis, which usually are reversible after discontinuation of the offending agent. These reversible reactions are not correlated with the risk of aplastic anemia, casting doubt on the effectiveness of routine monitoring of blood counts as a strategy to avoid aplastic anemia.

Because aplastic anemia is a rare event with drug use, it may occur because of an underlying metabolic or immunologic predisposition (gene polymorphism) in susceptible individuals. In the case of phenylbutazone-associated marrow aplasia, there is delayed oxidation and clearance of a related compound, acetanilide, as compared to either normal controls or those with aplastic anemia from other causes. This finding suggests excess accumulation of the drug as a potential mechanism for the aplasia. In some cases, drug interactions or synergy may be required to induce marrow aplasia. Cimetidine, a histamine H₂-receptor antagonist, is occasionally implicated in the onset of cytopenias and aplastic anemia, perhaps owing to a direct effect on early hematopoietic progenitor cells.⁵⁷ This drug accentuates the marrow-suppressive effects of the chemotherapy drug carmustine.⁵⁸ In several instances, it has been reported as a possible cause of marrow aplasia when given with chloramphenicol.

There appears to be little difference in the age distribution, gender, response to immunotherapy, marrow transplantation, or survival, whether or not a drug exposure preceded the onset of the marrow aplasia.

Toxic Chemicals

Benzene was the first chemical linked to aplastic anemia, based on studies in factory workers before the 20th century.^59-61 Benzene is used as a solvent and is employed in the manufacture of chemicals, drugs, dyes, and explosives. It has been a vital chemical in the manufacture of rubber and leather goods and has been used widely in the shoe industry, leading to an increased risk for aplastic anemia (and acute myelogenous leukemia) in workers exposed to a poorly regulated environment.⁶¹ In studies in China, aplastic anemia among workers was sixfold higher than in the general population.¹⁸

The U.S. Occupational Safety and Health Administration has lowered the permissible atmospheric exposure limit of benzene to 1 part per million (ppm) (8-hour time-weighted average) and short-term exposure to 5 ppm (15-minute time-weighted average). The National Institute for Occupational Safety and Health recommends limits of exposure of 0.1 ppm as the 8-hour weighted average and 1 ppm for 15-minute short-term exposure. Previous to that regulatory change, the frequency of aplastic anemia in workers exposed to greater than 100 ppm benzene was approximately 1 in 100 workers, which decreased to 1 in 1000 workers at 10 to 20 ppm exposure.⁶⁰

Organochlorine and organophosphate pesticide compounds have been suspected in the onset of aplastic anemia^62,63 and several studies have indicated an increased relative risk, especially for agricultural exposures^11,16,64,65 and household^11,65 exposures. These relationships are suspect because dose–disease relationships and other important factors have not been delineated, and several studies have not found an association with environmental exposures.^12,66 DDT (dichlorodiphenyltrichloroethane), lindane, and chlordane are insecticides that also have been associated with cases of aplastic anemia.^16,63 Occasional cases still occur following heavy exposure at industrial plants or after its use as a pesticide.⁶⁷ Lindane is metabolized in part to pentachlorophenol (PCP), another potentially toxic chlorinated hydrocarbon that is manufactured for use as a wood preservative. Cases of aplastic anemia and related blood disorders have been attributed to PCP over the past 25 years.^63,68 Prolonged exposures to petroleum distillates in the form of Stoddard solvent⁶⁹ and acute exposure to toluene through the practice of glue sniffing^70,71 also have been reported to cause marrow aplasia. Trinitrotoluene (TNT), an explosive used extensively during World Wars I and II, is absorbed readily by inhalation and through the skin.⁷² Fatal cases of aplastic anemia were observed in munitions workers exposed to TNT in Great Britain⁷³ from 1940 to 1946. In most cases, these conclusions have not been derived from specific studies but from accumulation of case reports or from patient histories, making conclusions provisional, although the argument for minimizing exposures to potential toxins is logical in any case.

Viruses

Non-A, -B, -C, -D, -E, -G Hepatitis Virus

A relationship between hepatitis and the subsequent development of aplastic anemia has been the subject of a number of case reports, and this association was emphasized by two major reviews in the 1970s.^74,75 In the aggregate, these reports summarized findings in more than 200 cases. In many instances, the hepatitis was improving or had resolved when the aplastic anemia was noted 4 to 12 weeks later. Approximately 10 percent of cases occurred more than 1 year after the initial diagnosis of hepatitis. Most patients were young (ages 18 to 20 years); two-thirds were male, and their survival was short (10 weeks). Although hepatitides A and B have been implicated in aplastic anemia in a small number of cases, most cases are related to non-A, non-B, non-C hepatitis.^76-78 Severe aplastic anemia developed in 9 of 31 patients who underwent liver transplantation for non-A, non-B, non-C hepatitis, but in none of 1463 patients transplanted for other indications.⁷⁹ Several lines of evidence indicate there is no causal association with hepatitis C virus, suggesting that an unknown viral agent is involved.^16,80,81 Hepatitis virus B or C can be a secondary infection, if carefully screened blood products are not used for transfusion. In 15 patients with posthepatitic aplastic anemia, no evidence was found for hepatitis A, B, C, D, E, or G, transfusion-transmitted virus, or parvovirus B19.⁸² Several reports suggest a relationship of parvovirus B19 to aplastic anemia,^83,84 whereas others have not.⁸¹ This relationship has not been established (Chap. 36). The effect of seronegative hepatitis may be mediated through an autoimmune T-cell effect because of evidence of T-cell activation and cytokine elaboration.²⁶ These patients also have a similar response to combined immunotherapy as do those with idiopathic aplastic anemia^85,86 (see “Treatment: Combination Immunotherapy” below).

Epstein-Barr Virus

Epstein-Barr virus (EBV) has been implicated in the pathogenesis of aplastic anemia.^87,88 The onset usually occurs within 4 to 6 weeks of infection. In some cases, infectious mononucleosis is subclinical, with a finding of reactive lymphocytes in the blood film and serologic results consistent with a recent infection (Chap. 82). EBV has been detected in marrow cells,⁸⁸ but it is uncertain whether marrow aplasia results from a direct effect or an immunologic response by the host. Patients have recovered following therapy with antithymocyte globulin.⁸⁸

Other Viruses

HIV infection frequently is associated with varying degrees of cytopenia. The marrow is often cellular, but occasional cases of aplastic anemia have been noted.^89-91 Marrow hypoplasia may result from viral suppression and from the drugs used to control viral replication in this disorder. Human herpes virus (HHV)-6 has caused severe marrow aplasia subsequent to marrow transplantation for other disorders.⁹²

Autoimmune Diseases

The incidence of severe aplastic anemia was sevenfold greater than expected in patients with rheumatoid arthritis.⁵⁴ It is uncertain whether the aplastic anemia is related directly to rheumatoid arthritis or to the various drugs used to treat the condition (gold salts, d-penicillamine, and nonsteroidal antiinflammatory agents). Occasional cases of aplastic anemia are seen in conjunction with systemic lupus erythematosus.⁹³ In vitro studies found either the presence of an antibody⁹⁴ or suppressor cell^95,96 directed against hematopoietic progenitor cells. Patients have recovered after plasmapheresis,⁹⁴ glucocorticoids,⁹⁶ or cyclophosphamide therapy,^95,97 which is compatible with an immune etiology.

Eosinophilic fasciitis, an uncommon connective tissue disorder with painful swelling and induration of the skin and subcutaneous tissue, has been associated with aplastic anemia.^98,99 Although it may be antibody-mediated in some cases, it has been largely unresponsive to therapy.⁹⁸ Nevertheless, (1) stem cell transplantation, (2) immunosuppressive therapy using cyclosporine, (3) immunosuppressive therapy using antithymocyte globulin (ATG), or (4) immunosuppressive therapy with ATG and cyclosporine has cured or significantly ameliorated the disease in a few patients.^98,99

Severe aplastic anemia also has been reported coincident with immune thyroid disease (Graves disease)^100-104 and the aplasia has been reversed with treatment of the hyperthyroidism. Aplastic anemia has occurred in association with thymoma.^104-110 Autoimmune renal disease and aplastic anemia have occurred concurrently. The underlying relationship may be the role of cytotoxic T lymphocytes in the pathogenesis of several autoimmune diseases and in aplastic anemia.¹¹¹

Pregnancy

There are a number of reports of pregnancy-associated aplastic anemia, but the relationship between the two conditions is not always clear.^112-117 In some patients, preexisting aplastic anemia is exacerbated with pregnancy, only to improve following termination of the pregnancy.^112,113 In other cases, the aplasia develops during pregnancy with recurrences during subsequent pregnancies.^113,114 Termination of pregnancy or delivery may improve the marrow function, but the disease may progress to a fatal outcome even after delivery.^112-114 Therapy may include elective termination of early pregnancy, supportive care, immunosuppressive therapy, or marrow transplantation after delivery. Pregnancy in women previously treated with immunosuppression for aplastic anemia can result in the birth of a normal newborn.¹¹⁷ In this latter study of 36 pregnancies, 22 were uncomplicated, 7 were complicated by a relapse of the marrow aplasia, and 5 without marrow aplasia required red cell transfusion during delivery.¹¹⁷ One death occurred from cerebral thrombosis in a patient with paroxysmal nocturnal hemoglobinuria (PNH) and marrow aplasia.

Iatrogenic Causes

Although marrow toxicity from cytotoxic chemotherapy or radiation produces direct damage to stem cells and more mature cells, resulting in marrow aplasia, most patients with acquired aplastic anemia cannot relate an exposure that would be responsible for marrow damage.

Chronic exposure to low doses of radiation or use of spinal radiation for ankylosing spondylitis is associated with an increased, but delayed, risk of developing aplastic anemia and acute leukemia.^118,119 Patients who were given thorium dioxide (Thorotrast) as an intravenous contrast medium suffered numerous late complications, including malignant liver tumors, acute leukemia, and aplastic anemia.¹²⁰ Chronic radium poisoning with osteitis of the jaw, osteogenic sarcoma, and aplastic anemia was seen in workers who painted watch dials with luminous paint when they moistened the brushes orally.¹²¹

Acute exposures to large doses of radiation are associated with the development of marrow aplasia and a gastrointestinal syndrome.^122,123 Total-body exposure to between 1 and 2.5 Gy leads to gastrointestinal symptoms and depression of leukocyte counts, but most patients recover. A dose of 4.5 Gy leads to death in half the individuals (LD₅₀) owing to marrow failure. Higher doses in the range of 10 Gy are universally fatal unless the patient receives extensive supportive care followed by marrow transplantation. Aplastic anemia associated with nuclear accidents was seen after the disaster that occurred at the Chernobyl nuclear power station in the Ukraine in 1986.¹²⁴

Antineoplastic drugs such as alkylating agents, antimetabolites, and certain cytotoxic antibiotics have the potential for producing marrow aplasia. In general, this is transient, is an extension of their pharmacologic action, and resolves within several weeks of completing chemotherapy. Although unusual, severe marrow aplasia can follow use of the alkylating agent, busulfan, and may persist indefinitely. Patients may develop marrow aplasia 2 to 5 years after discontinuation of alkylating agent therapy. These cases often evolve into hypoplastic myelodysplastic syndromes.

Stromal Microenvironment and Growth Factors

Short-term clonal assays for marrow stromal cells have shown variable defects in stromal cell function in patients with aplastic anemia. Serum levels of stem cell factor (SCF) have been either moderately low or normal in several studies of aplastic anemia.^125,126 Although SCF augments the growth of hematopoietic colonies from aplastic anemia patient’s marrows, its use in patients has not led to clinical remissions. Another early acting growth factor, FLT-3 ligand, is 30- to 100-fold elevated in the serum of patients with aplastic anemia, although the pathobiologic effect of this change is unclear.¹²⁷ Fibroblasts grown from patients with severe aplastic anemia have subnormal cytokine production. However, serum levels of granulocyte colony-stimulating factor,¹²⁸ erythropoietin,¹²⁹ and thrombopoietin (TPO)¹³⁰ are usually high. Synthesis of IL-1, an early stimulator of hematopoiesis, is decreased in mononuclear cells from patients with aplastic anemia.¹³¹ Studies of the microenvironment have shown relatively normal stromal cell proliferation and growth factor production.¹³² These findings, coupled with the limited response of patients with aplastic anemia to growth factors, suggest that cytokine deficiency is not the etiologic problem in most cases. The most compelling argument is that most patients transplanted for aplastic anemia are cured with allogeneic donor stem cells and autologous stroma.¹³³

A rare exception to the negligible pathogenetic role of hematopoietic growth factors in the etiology of aplastic anemia is the homozygous or mixed heterozygous mutation of the TPO receptor gene, MPL, which can cause amegakaryocytic thrombocytopenia that evolves, later, into aplastic anemia (Chap. 117). Furthermore, eltrombopag, a TPO receptor agonist, can stimulate mono, or in some patients, bilineage or trilineage recovery of blood counts that may be sustained off therapy (see “Treatment: Cytokines” below).

CLINICAL FEATURES

The onset of symptoms of aplastic anemia may be gradual with pallor, weakness, dyspnea, and fatigue as a result of the anemia. Dependent petechiae, bruising, epistaxis, vaginal bleeding, and unexpected bleeding at other sites secondary to thrombocytopenia are frequent presenting signs of the underlying marrow disorder. Rarely, it may be more dramatic with fever, chills, and pharyngitis or other sites of infection resulting from severe neutropenia and monocytopenia. Physical examination generally is unrevealing except for evidence of anemia (e.g., conjunctival and cutaneous pallor, resting tachycardia) or cutaneous bleeding (e.g., ecchymoses and petechiae), gingival bleeding and intraoral purpura. Lymphadenopathy and splenomegaly are not features of aplastic anemia; such findings suggest an alternative diagnosis such as a clonal myeloid or lymphoid disease.

LABORATORY FEATURES

Blood Findings

Patients with aplastic anemia have varying degrees of pancytopenia. Anemia is associated with a low reticulocyte index. The reticulocyte count is usually less than 1 percent and may be zero despite the high levels of erythropoietin. Absolute reticulocyte counts are usually fewer than 40,000/μL (40 × 10⁹/L). Macrocytes may be present in the blood film and the mean cell volume (MCV) increased. The absolute neutrophil and monocyte count are low. An absolute neutrophil count fewer than 500/μL (0.5 × 10⁹/L) along with a platelet count fewer than 30,000/μL (30 × 10⁹/L) is indicative of severe disease, and a neutrophil count below 200/μL (0.2 × 10⁹/L) denotes very severe disease (see Table 35–1). Lymphocyte production is thought to be normal, but patients may have mild lymphopenia. Platelets function normally. Significant qualitative changes of red cell, leukocyte, or platelet morphology on the blood film are not features of classical acquired aplastic anemia. On occasion, only one cell line is depressed initially, which may lead to an early diagnosis of pure red cell aplasia or amegakaryocytic thrombocytopenia. In such patients, other cell lines will fail shortly thereafter (days to weeks) and permit a definitive diagnosis. Table 35–4 is a plan for the initial laboratory investigation.

Plasma Findings

The plasma contains high levels of hematopoietic growth factors, including erythropoietin, TPO, and myeloid colony-stimulating factors. Growth factor levels need not be measured, however, for clinical care. Plasma iron values are usually high, and ⁵⁹Fe clearance is prolonged, with decreased incorporation into red cells.

Marrow Findings

Morphology

The marrow aspirate typically contains numerous spicules with empty, fat-filled spaces, and relatively few hematopoietic cells. Lymphocytes, plasma cells, macrophages, and mast cells may be present. On occasion, occasional spicules are cellular or even hypercellular (“hot spots”), but megakaryocytes usually are reduced. These focal areas of residual hematopoiesis do not appear to be of prognostic significance. Residual granulocytic cells generally appear normal, but it is not unusual to see mild macronormoblastic erythropoiesis, presumably as a result of the high levels of erythropoietin. Marrow biopsy is essential to confirm the overall hypocellularity (Fig. 35-2), as a poor yield of spicules and cells occurs in marrow aspirates in other disorders, especially if fibrosis is present.

In severe aplastic anemia, as defined by the International Aplastic Anemia Study Group, less than 25 percent cellularity or less than 50 percent cellularity with less than 30 percent hematopoietic cells is seen in the marrow.

Progenitor Cell Growth

In vitro CFU-GM and BFU-E colony assays reveal a marked reduction in progenitor cells.^19-22

Cytogenetic and Genetic Studies

Cytogenetic analysis may be difficult to perform owing to low cellularity; thus, multiple aspirates may be required to provide sufficient cells for study. The results are normal in aplastic anemia. Clonal cytogenetic abnormalities in otherwise apparent aplastic anemia is indicative of an underlying hypoplastic clonal myeloid disease.¹³⁴ The move to newer techniques such as microarray-based comparative genomic hybridization (CGH) permits detection of aneuploidies, deletions, duplications, and/or amplifications of any locus represented on an array. In addition, microarray-based CGH is an effective tool for the detection of submicroscopic chromosomal abnormalities. This approach would increase the sensitivity to detect chromosome abnormalities in very hypocellular marrow samples, compared to standard G-banding, despite dilution of scant hematopoietic cells with nonhematopoietic stromal cells (e.g., fibroblasts). Next-generation sequencing of targeted exons has uncovered 32 mutations associated with myeloid malignancies. These mutations occurred in nearly 20 percent (29 of 150 patients) of cases of aplastic anemia. These mutations include the genes ASXL1, DNMT3A, and BCOR, which are considered driver mutations in myelodysplastic syndrome and acute myelogenous leukemia. Seventeen of the 29 patients with one of these three mutations evolved to overt myelodysplasia.¹³⁵

Imaging Studies

Magnetic resonance imaging (MRI) can be used to distinguish between marrow fat and hematopoietic cells.¹³⁶ This approach may be a more useful overall estimate of marrow hematopoietic cell density than morphologic techniques and may help differentiate hypoplastic myelogenous leukemia from aplastic anemia.¹³⁰

DIFFERENTIAL DIAGNOSIS

Any disease that can present with pancytopenia may mimic aplastic anemia if only the blood counts are considered. Measurement of the reticulocyte count and an examination of the blood film and marrow biopsy are essential early steps to arrive at a diagnosis. A reticulocyte percentage of 0.5 percent to zero is strongly indicative of aplastic erythropoiesis, and when coupled with leukopenia and thrombocytopenia, points to aplastic anemia. Absence of qualitative abnormalities of cells on the blood film and a markedly hypocellular marrow are characteristic of acquired aplastic anemia. The disorders most commonly confused with severe aplastic anemia include the approximately 5 to 10 percent of patients with myelodysplastic syndromes who present with a hypoplastic rather than a hypercellular marrow. Myelodysplasia should be considered if there is abnormal blood film morphology consistent with myelodysplasia (e.g., poikilocytosis, basophilic stippling, neutrophils with hypogranulation or the pseudo–Pelger-Hüet anomaly). Marrow erythroid precursors in myelodysplasia may have dysmorphic features. Pathologic sideroblasts are inconsistent with aplastic anemia and a frequent feature of myelodysplasia. Granulocyte precursors may have reduced or abnormal granulation. Megakaryocytes may have abnormal nuclear lobulation (e.g., unilobular micromegakaryocytes; Chap. 87). If clonal cytogenetic abnormalities are found, a clonal myeloid disorder, especially myelodysplastic syndrome or hypocellular myelogenous leukemia is likely. MRI studies of bone may be useful in differentiating severe aplastic anemia from clonal myeloid syndromes. The former gives a fatty signal and the latter a diffuse cellular pattern.

A hypocellular marrow frequently is associated with PNH. PNH is characterized by an acquired mutation in the PIG-A gene that encodes an enzyme that is required to synthesize mannolipids. The gene mutation prevents the synthesis of the glycosylphosphatidylinositol anchor precursor. This moiety anchors several proteins, including inhibitors of the complement pathway to blood cell membranes, and its absence accounts for the complement-mediated hemolysis in PNH. As many as 50 percent of patients with otherwise typical aplastic anemia have evidence of glycosylphosphatidylinositol molecule defects and diminished phosphatidylinositol-anchored protein on leukocytes and red cells as judged by flow cytometry, analogous to that seen in PNH.¹³⁷ The decrease or absence of these membrane proteins may make the PNH clone of cells resistant to the acquired immune attack on normal marrow components, or the phosphatidylinositol-anchored protein(s) on normal cells provides an epitope that initiates an aberrant T-cell attack, leaving the PNH clone relatively resistant (Chap. 40).²⁶

Occasionally, apparent aplastic anemia may be the prodrome to childhood¹³⁸ or, less commonly, adult¹³⁹ acute lymphoblastic leukemia. Sometimes, careful examination of marrow cells by light microscopy or flow cytometry will uncover a population of leukemic lymphoblasts. In other cases, the acute leukemia may appear later. Hairy-cell leukemia, Hodgkin disease, or another lymphoma subtype, rarely, may be preceded by a period of marrow hypoplasia. Immunophenotyping of marrow and blood cells by flow cytometry for CD25 may uncover the presence of hairy cells. Other clinical features may be distinctive (Chap. 93). Organomegaly such as lymphadenopathy, hepatomegaly, or splenomegaly are inconsistent with the atrophic (hypoproliferative) features of aplastic anemia. Large granular lymphocytic leukemia has also been associated with aplastic anemia. Rare cases of typical acquired aplastic anemia have been followed by t(9;22)-positive acute lymphocytic leukemia (ALL) or chronic myelogenous leukemia (CML).¹³⁹

RELATIONSHIP AMONG APLASTIC ANEMIA, PAROXYSMAL NOCTURNAL HEMOGLOBINURIA, AND CLONAL MYELOID DISEASES

In addition to the diagnostic difficulties occasionally presented by patients with hypoplastic myelodysplastic syndromes, hypoplastic acute myelogenous leukemia (AML), or PNH with hypocellular marrows, there may be a more fundamental relationship among these three diseases and aplastic anemia. The development of clonal cytogenetic abnormalities such as monosomy 7 or trisomy 8 in a patient with aplastic anemia portends the evolution of a myelodysplastic syndrome or acute leukemia. Occasionally, these cytogenetic markers have been transient, and in cases with disappearance of monosomy 7, hematologic improvement has occurred as well.¹⁴⁰ Persistent monosomy 7 carries a poor prognosis as compared to trisomy 8.^141,142

As many as 20 percent of patients with aplastic anemia have a 5-year probability of developing myelodysplasia.¹⁴⁰ If one excludes any transformation to a clonal myeloid disorder that occurs up to 6 months after treatment to avoid misdiagnosis among the hypoplastic clonal myeloid diseases, the frequency of a clonal myeloid disorder was nearly 15 times greater in patients treated with immunosuppression as compared to those treated with marrow transplantation after 39 months of observation.¹⁴³ This finding suggests either that immune suppression by anti–T-cell therapy enhances the evolution of a neoplastic clone or that it does not suppress the intrinsic tendency of aplastic anemia to evolve to a clonal disease, but provides the increased longevity of the patient required to express that potential. The latter interpretation is more likely as patients successfully treated solely with androgens develop clonal disease as frequently as those treated with immunosuppression.¹⁴⁴ Transplantation may reduce the potential to clonal evolution in patients with aplastic anemia by reestablishing robust lymphohematopoiesis.

Telomere shortening also may play a pathogenetic role in the evolution of aplastic anemia into myelodysplasia. Patients with aplastic anemia have shorter telomere lengths than matched controls, and patients with aplastic anemia with persistent cytopenias had greater telomere shortening over time than matched controls. Three of five patients with telomere lengths less than 5 kb developed clonal cytogenetic changes, whereas patients with longer telomeres did not develop such diseases.^23,145

The findings of mutated genes considered driver mutations in myelodysplastic syndrome or AML (see “Marrow Findings: Cytogenetic and Genetic Studies” earlier) in nearly 20 percent of a population of patients with clinical aplastic anemia indicates that clonal hematopoiesis may develop or be present surreptitiously. The precise relationships to the aplastic anemia lesion is uncertain but could be caused the outgrowth of a clone of cells in the background of severally suppressed polyclonal hematopoietic stem cells. These findings were more common in patients with a long duration of disease and with shorter telomeres.¹³⁵

The relationship of PNH to aplastic anemia remains enigmatic. Because hematopoietic stem cells lacking the phosphatidylinositol-anchored proteins are present in many or all normal persons in very small numbers,¹⁴⁶ it is not surprising that more than 50 percent of patients with aplastic anemia may have a PNH cell population as detected by immunophenotyping.¹³⁷ The probability of patients with aplastic anemia developing a clinical syndrome consistent with PNH is 10 to 20 percent, and this is not a consequence of immunosuppressive treatment.¹⁴⁰ Patients also may present with the hemolytic anemia of PNH and later develop progressive marrow failure so that any pathogenetic explanation should consider both types of development of aplastic marrows in PNH. The PIG-A mutation may confer either a proliferative or survival advantage to PNH cells.^147,148 A survival advantage could result if the anchor protein or one of its ligands served as an epitope for the T-lymphocyte cytotoxicity, which induces the marrow aplasia. In this case, the presenting event could either reflect cytopenias or the sensitivity of red cells to complement lysis and hemolysis, depending on the intrinsic proliferative potential of the PNH clone.

Within our current state of knowledge, aplastic anemia is an autoimmune process, and any residual hematopoiesis is presumably polyclonal. This is a critical distinction from hypoplastic leukemia and PNH, which are clonal (neoplastic) diseases. The environment of the aplastic marrow, however, may favor the eventual evolution of a mutant (malignant) clone, especially if immunotherapy is used, whereas hematopoietic stem cell transplantation may either ablate threatening minor clones or establish more robust hematopoiesis, an environment less conducive to clonal evolution.

TREATMENT

Approach to Therapy

Severe anemia, bleeding from thrombocytopenia, and, uncommonly at the time of diagnosis, infection secondary to granulocytopenia and monocytopenia require prompt attention to remove potential life-threatening conditions and improve patient comfort (Table 35–5). More specific treatment of the marrow aplasia involves two principal options: (1) syngeneic or allogeneic hematopoietic stem cell transplantation or (2) combination immunosuppressive therapy with ATG and cyclosporine. The selection of the specific mode of treatment depends on several factors, including the patient’s age and condition and the availability of a suitable allele-level HLA-matched hematopoietic stem cell donor. In general, transplantation is the preferred treatment for children and most otherwise healthy younger adults. Early histocompatibility testing of siblings is of particular importance because it establishes whether there is an optimal donor available to the patient for transplantation. The preferred stem cell source is a histocompatible sibling matched at the HLA-A, -B, -C, and -DR loci.

Supportive Care

The Use of Blood Products

Although it has been recommended that red cell and platelet transfusions be used sparingly in potential transplant recipients to minimize sensitization to histocompatibility antigens, this has become less important since ATG and cyclophosphamide have been used as the preparative regimen for transplantation in aplastic anemia, as their use has markedly reduced the problem of graft rejection.¹⁴⁹

Cytomegalovirus (CMV)-reduced risk red cells and platelets should be given to a potential transplant recipient to minimize problems with CMV infections after transplantation. Once a patient is shown to be CMV-positive, this restriction is no longer necessary. Leukocyte-depletion filters or CMV serotesting are equivalent methods of decreasing the risk of transmitting CMV.

Red Cell Transfusion

Packed red cells to alleviate symptoms of anemia usually are indicated at hemoglobin values below 8 g/dL (80 g/L), unless comorbid medical conditions require a higher hemoglobin concentration. These products should be leukocyte-depleted to lessen leukocyte and platelet sensitization and to reduce subsequent transfusion reactions and radiated to reduce the potential for a transfusion-related graft-versus-host reaction. It is important not to transfuse patients with red cells (or platelets) from family members if transplantation within the family is remotely possible, as this approach may sensitize patients to minor histocompatibility antigens, increasing the risk of graft rejection after marrow transplantation. Following a marrow transplant, or in those individuals in whom transplantation is not a consideration, family members may be ideal donors for platelet products. Because each unit of red cells adds approximately 200 mg of iron to the total body iron, over the long-term transfusion-induced iron overload may occur. This is not a major problem in patients who respond to transplantation or immunosuppressive therapy, but it is an issue in nonresponders who require continued transfusion support. In the latter case, consideration should be given to iron-chelation therapy. Newer oral agents make this procedure easier to effect (Chap. 48).¹⁵⁰

Platelet Transfusion

It is important to assess the risk of bleeding in each patient. Most patients tolerate platelet counts of 10,000/μL (10 × 10⁹/L) without undue bruising or bleeding, unless a systemic infection is present or vascular integrity is impaired.^151,152 A traumatic injury or surgery requires transfusion to greater than 50,000/μL or greater than 100,000/μL, respectively. Administration of ε-aminocaproic acid, 50 mg/kg per dose every 4 hours orally or intravenously, may reduce the bleeding tendency.¹⁵³ Pooled random-donor platelets may be used until sensitization ensues, although it is preferable to use single-donor platelets from the onset to minimize sensitization to HLA or platelet antigens. Subsequently, single-donor apheresis products or HLA-matched platelets may be required.

Platelet refractoriness is a major problem with long-term transfusion support.¹⁵⁴ This may occur transiently, with fever or infection, or as a chronic problem secondary to HLA sensitization. In the past, this occurred in approximately 50 percent of patients after 8 to 10 weeks of transfusion support. Filtration of blood and platelet concentrates to remove leukocytes reduces this problem to approximately 15 percent of patients receiving chronic transfusions.^154,155 Patient’s should also get ABO-identical platelets because this enhances platelet survival and further decreases refractoriness to platelet transfusion. Single-donor HLA-matched apheresis-harvested platelets may be necessary in previously pregnant or transfused patients who are already allosensitized or who so become after treatment with leukoreduced platelets. The frequency of either of these events is less than 10 percent. Chapter 139 discusses approaches to chronic platelet transfusion.

Management of Neutropenia

Neutropenic precautions should be applied to hospitalized patients with a severe depression of the neutrophil count. The level of neutrophils requiring precautions is fewer than 500/μL (0.5 × 10⁹/L). One approach is to use private rooms, with requirements for face masks and handwashing with antiseptic soap. Unwashed fresh fruits and vegetables should be avoided as they are sources of bacterial contamination. It is uncommon for patients with aplastic anemia to present with a significant infection. When patients with aplastic anemia become febrile, cultures should be obtained from the throat, sputum (if any), blood, urine, stool, and any suspicious lesions. Broad-spectrum bactericidal antibiotics should be initiated promptly, without awaiting culture results. The choice of antibiotics depends on the prevalence of organisms and their antibiotic sensitivity in the local setting. Organisms of concern usually include Staphylococcus aureus (notably methicillin-and oxacillin-resistant strains), Staphylococcus epidermidis (in patients with venous access devices), and Gram-negative organisms. Patients with persistent culture-negative fevers should be considered for antifungal treatment (Chap. 24).

In the past, leukocyte transfusions were used on a daily basis to reduce the short-term mortality from infections. It was unusual to detect more than 100 to 200 neutrophils per microliter for more than a few hours after transfusion. The yield of neutrophils can be increased by administering granulocyte colony-stimulating factor (G-CSF) to the donor,¹⁵⁶ but most physicians avoid using white cell products because present-day antibiotics are usually sufficient to treat a patient for an episode of sepsis. Notable exceptions include documented invasive aspergillosis unresponsive to amphotericin (particularly in the posttransplant setting), infections with organisms resistant to all known antibiotics, and when blood cultures remain positive in spite of antibiotic treatment. Leukocyte transfusion is more effective in children and adults with smaller body size, as transfused leukocytes have a smaller distribution space, which results in higher blood and tissue concentrations.

Specific Treatment

Hematopoietic Stem Cell Transplantation

Prompt therapy usually is indicated for patients with severe aplastic anemia. The major curative approach is hematopoietic stem cell transplantation from a histocompatible sibling.^157-159 Chapter 23 describes this treatment modality. Only 20 to 30 percent of patients in the United States have compatible sibling donors (related to average family size). In the unusual case of an identical twin donor, conditioning is required to obliterate the immune disease in the recipient, but it can be limited to cyclophosphamide. In this setting, an 80 to 90 percent survival is expected. Marrow stem cells seem to perform better than blood stem cells when used as a source for patients with aplastic anemia, although this is under continued study. The results of transplantation are best in patients younger than age 20 years (80 to 90 percent long-term survival) but decrease every decade of increasing age thereafter. Posttransplant mortality is increased and survival decreased with increasing age (Fig. 35-3). In patients older than age 40 years, survival in matched sibling transplant is reduced to approximately 50 percent.¹⁶⁰ There are still uncertainties about the optimal conditioning program in younger and older patients. ATG, cyclophosphamide, total-body radiation, fludarabine, and alemtuzumab are among the agents being studied.^157,159-161 Alemtuzumab-containing regimens appear to improve outcome by decreasing the frequency of chronic graft-versus-host disease, which could make it useful in older patients.^{161,161a,161b}

The longer the delay between diagnosis and transplantation, the less likely is a salutary outcome, probably as a result of a greater number of transfusions and a higher likelihood of pretransplantation infection. Acute and chronic graft-versus-host disease are serious complications, and therapy to prevent or ameliorate them is a standard part of posttransplantation treatment.^157,160 Transplantations have been performed using stem cells from partially matched siblings or unrelated, histocompatible donors recruited through the National Marrow Donor Program or similar organizations in other countries.¹⁶² Umbilical cord blood is an alternative source of stem cells from unrelated donors (or, rarely, siblings) for transplantation in children, but the results are optimal with matched sibling transplantation. Alternatively, the use of high-resolution, HLA typing of a matched, unrelated donor markedly improves the prognosis for transplantation.¹⁶³ High-resolution DNA matching at HLA-A, -B, -C, and -DRB1 (8 of 8 alleles) is considered the lowest level of matching consistent with the highest level of survival. If there is an HLA mismatch at one or more loci, especially HLA-A or -DRB1, the outcome is compromised,¹⁶³ and immunosuppression with combined therapy may be preferred initially, depending on patient age, CMV status, and disease severity. Older patients have a much lower favorable response with alternative, non–matched-sibling, donor transplantations. The use of hematopoietic stem cell transplantation can be considered for patients who do not respond or who no longer respond to immunotherapy.¹⁶⁰ If the patient in question is a candidate for stem cell transplantation based on all relevant factors, transplantation could be considered at any age for a patient with a syngeneic donor; transplantation could be considered as a first-choice therapy up to age 50 years for a patient with an HLA allele-level matched sibling donor; and transplantation could be considered a first-choice therapy if an allele-level HLA-matched unrelated donor is available for patients younger than age 20 years.¹⁶⁰ These guidelines are subject to the unique or special circumstances of an individual case. For example, if patients with aplastic anemia undergo gene sequencing and a mutation known to be a driver mutation for myelodysplasia or AML is found, allogeneic hematopoietic stem cell transplantation may prove to be a preferred approach.

Components of Anti–T-Lymphocyte (Immunosuppressive) Therapy

Antilymphocyte Serum and Antithymocyte Globulin

ATG and ALG act principally by reducing cytotoxic T cells. This involves ATG-induced apoptosis through both FAS and TNF pathways.¹⁶⁴ Cathepsin B also plays a role in T-cell cytotoxicity at clinical concentrations of ATG, but may involve an independent apoptosis pathway.¹⁶⁵ ATG and ALG also release hematopoietic growth factors from T cells.^166,167 Horse and rabbit ATG are licensed in the United States. Skin tests against horse serum should be performed prior to administration.¹⁶⁸ If positive, the patient may be desensitized. ATG therapy is given daily for 4 to 10 days with doses of 15 to 40 mg/kg. Fever and chills are common during the first day of treatment. Concomitant treatment with glucocorticoids, such as methylprednisolone or dexamethasone lessens the reaction to ATG. Several studies have compared equine to rabbit ATG in the immunotherapy of aplastic anemia, contemporaneously or using historical comparisons. The consensus is that equine ATG is superior to rabbit and, if available, is recommended as the first line of therapy (Table 35–6).^169-177 Nevertheless, rabbit ATG is effective and should be considered if equine ATG does not result in a satisfactory outcome (Fig. 35-4).

ATG treatment may accelerate platelet destruction, reduce the absolute neutrophil count, and cause a positive direct antiglobulin (Coombs) test. This effect may lead to an increase in transfusion requirements during the 4- to 10-day treatment interval. Serum sickness, characterized by spiking fevers, skin rashes, and arthralgias, occurs commonly 7 to 10 days from the first dose. The clinical manifestations of serum sickness can be diminished by increasing the glucocorticoid dose from day 10 to day 17 after treatment. Approximately one-third of patients no longer require transfusion support after treatment with ATG alone.^178-180

Of 358 patients responding to immunosuppressive therapy, principally ATG alone, 74 (21 percent) relapsed after a mean of 2.1 years. The actuarial incidence of relapse was 35 percent at 10 years.¹⁸¹ Similar results were observed when 227 patients were treated with immunosuppression, primarily ATG alone.¹⁸² The actuarial survival at 15 years was 38 percent following immunosuppression.¹⁸¹ However, a combination of immunosuppressive agents provides more effective therapy than ATG alone (see “Combination Immunotherapy” below).

Twenty-eight (22 percent) of 129 patients treated with ALG developed myelodysplasia, leukemia, PNH, or combined disorders.¹⁸³ This tendency to relapse and to develop clonal hematologic disorders was reviewed by the European Cooperative Group for Bone Marrow Transplantation in 468 patients, most of whom received ATG.¹⁸⁴ The risk of a hematologic complication increased continuously and reached 57 percent at 8 years after immunosuppressive therapy. A further survey found 42 (5 percent) malignancies in 860 patients treated with immunosuppression, whereas only 9 (1 percent) malignancies were seen in 748 patients who received marrow transplants.¹⁸⁵

There are no predictors that augur the risk of clonal evolution in an individual patient, although shorter telomere length at diagnosis and poorer prognosis are associated.¹⁸⁶

Cyclosporine

Administration of cyclosporine, a cyclic polypeptide that inhibits IL-2 production by T lymphocytes and prevents expansion of cytotoxic T cells in response to IL-2, is another approach to immunotherapy. After the initial report of its ability to induce remission in 1984,¹⁸⁷ several groups have used cyclosporine as either (1) primary treatment,^188-191 (2) in patients refractory to ATG or glucocorticoids,^189-194 (3) in combination with G-CSFs,^195,196 or (4) in varying combinations with other modes of therapy.¹⁹⁷ Cyclosporine is administered orally at 10 to 12 mg/kg per day for at least 4 to 6 months. Dosage adjustments may be required to maintain trough blood levels of 200 to 400 ng/mL. Renal impairment is common and may require increased hydration or dose adjustments to keep creatinine values below 2 mg/dL. Cyclosporine also may cause moderate hypertension, a variety of neurologic manifestations, and other side effects. Several drug classes interact with cyclosporine to either increase (e.g., some antibiotics and antifungals) or decrease (e.g., some anticonvulsants) blood levels. Responses usually are seen by 3 months and may range from achieving transfusion independence to complete remission. Approximately 25 percent of patients respond to this agent when used alone, but the response rate has ranged from 0 to 80 percent in various reports.¹⁹⁷

Although immunosuppression with ALG or ATG has been used the longest and has a seemingly better response rate, there are certain advantages to cyclosporine. This drug does not require hospitalization or use of a central venous catheter. Fewer platelet transfusions are required during the first few weeks of therapy compared to treatment with ALG or ATG. A French cooperative trial showed equal effectiveness of cyclosporine compared to ATG plus prednisone.¹⁹⁸ In this crossover study of newly diagnosed patients, survival of approximately 65 percent was observed 12 months after diagnosis.

Combination Immunotherapy

Combination treatment of severe aplastic anemia usually includes, for example, ATG, 40 mg/kg per day, for 4 days; cyclosporine, 10 to 12 mg/kg per day, for 6 months and methylprednisolone, 1 mg/kg per day, for 2 weeks.¹⁹⁹ The dose of cyclosporine is adjusted to maintain a trough level of 200 to 400 ng/mL. Prophylaxis for Pneumocystis carinii with daily trimethoprim-sulfamethoxazole or with monthly pentamidine inhalations should be considered for these patients as they receive immunosuppressive therapy.

The addition of cyclosporine to the combination of ALG and glucocorticoids improves response rates to approximately 70 percent of patients (Table 35–7).^200,201 G-CSF added to the combined immunosuppressive therapy does not increase response rate or survival.²⁰² Response is usually defined as a significant improvement in red cells, white cells, and platelets to eliminate risk of infection and bleeding and the requirement for red cell transfusions.

The 5-year survival after completion of combination immunosuppressive therapy may approximate that after stem cell transplantation.²⁰⁴ Forty-eight children treated between 1983 and 1992 had a 10-year survival of approximately 75 percent for marrow transplantation and approximately 75 percent for combined immunosuppressive therapy, although there were only half the number of severely affected patients in the immunosuppressive therapy group.²⁰⁵ Thus, immunosuppression may be preferable for patients who are older than 30 years of age and in those who may experience a delay in finding a suitable donor. Marrow transplants are, however, curative for aplastic anemia, whereas more frequent sequelae have been found after immunosuppressive therapy,^206-208 notably a substantial rate of evolution to a myelodysplastic syndrome or AML.

A National Institutes of Health protocol was designed to increase immune tolerance by specific deletion of activated T lymphocytes that target primitive hematopoietic progenitor cells.²⁶ Concurrent administration of cyclosporine with ATG may diminish the ATG effect so that in this program cyclosporine is introduced at a later time. The addition of new immunosuppressive agents, such as mycophenolate mofetil, rapamycin, or monoclonal antibodies, to the IL-2 receptor may be more effective in decreasing cytotoxic T cells, sparing the targeted hematopoietic stem cells.²⁶

For the 30 to 40 percent of patients who relapse after immunotherapy, retreatment with ATG and cyclosporine is effective in 50 to 60 percent of them.^209,210 Alternatively, alemtuzumab, a monoclonal anti-CD52 antibody that targets that antigen on T lymphocytes, has been an effective immunosuppressive agent in relapsed and in refractory patients, and it may be administered with cyclosporine.^{211–213,213a}

Combination of Immunotherapy and Eltrombopag

Approximately one-quarter to one-third of patients with acquired aplastic anemia who are treated with ATG and cyclosporine fail to restore hematopoiesis and do not achieve reasonable blood cell counts. When eltrombopag was combined with ATG and cyclosporine and administered to previously untreated patients with severe aplastic anemia, the response rate was 94 percent at 6 months in the group treated from day 1 with all three agents, compared with a 66 percent response rate in historical controls treated with ATG and cyclosporine.^213b The overall survival of patients treated with the three agents at 2 years was 97 percent. This triple drug approach has not yet been studied widely or for a long follow-up period, but the improvement over ATG and cyclosporine is so striking that it is likely to become the treatment of choice in patients not amenable to allogeneic sibling transplantation as a first treatment option (i.e., young age and histocompatible sibling donor). Evolution to a clonal myeloid disorder occurred in 8 percent, similar to historical controls treated with ATG and cyclosporine without the addition of eltrombopag.

Other Approaches to Immunotherapy

Alemtuzumab

Alemtuzumab is a humanized IgG1 monoclonal antibody directed against the CD52 protein; CD52 is expressed on all lymphocytes and monocytes. Alemtuzumab produces profound and persistent lymphopenia. The antibody has been used to treat a severe aplastic anemia that does not respond to or relapses after use of horse antithymocyte globulin.^{211–213,213a} It also has been used as a conditioning agent prior to allogeneic hematopoietic stem cell transplantation.^{161,161a,161b}

High-Dose Glucocorticoid Treatment

Marrow recovery can occur after very high doses of glucocorticoids.^214,215 Methylprednisolone in the range of 500 to 1000 mg daily for 3 to 14 days has been successful, but the side effects, which include marked hyperglycemia and glycosuria, electrolyte disturbances, gastric irritation, psychosis, increased infections, and aseptic necrosis of the hips, can be severe. Glucocorticoids at lower doses commonly are used only as a component of combination therapy for aplastic anemia to ameliorate the toxic effects of ATG and in providing additional lymphocyte suppression.

High-Dose Cyclophosphamide Therapy

High-dose cyclophosphamide has been used as a form of immunosuppression.²¹⁶ Although it would seem inappropriate to administer high doses of chemotherapy to patients with severe marrow aplasia, this approach was based on observations of autologous recovery after preparative therapy for allogeneic transplants not followed by a transplantation.⁶ In an early study, 10 patients who received cyclophosphamide at 45 mg/kg per day intravenously for 4 days with or without cyclosporine for an additional 100 days had gradual neutrophil and platelet recovery over 3 months. Seven patients responded completely and remained in remission 11 years after treatment. High-dose cyclophosphamide treatment may spare hematopoietic stem cells, which have high levels of aldehyde dehydrogenase and are relatively resistant to cyclophosphamide.^217,218 Thus, cyclophosphamide in this situation may be more immunosuppressive than myelotoxic. The most extensive trial of high-dose cyclophosphamide resulted in 65 percent of patients responding completely at 50 months.²¹⁹ However, the role of this regimen as initial therapy is not clear because of early toxicity that may exceed that of the ATG and cyclosporine combination.²²⁰ The probability of a durable remission may be superior, but there are insufficient data (comparative clinical trials) to conclude whether high-dose cyclophosphamide provides better long-term results than ATG and cyclosporine. The latter approach is favored at this time.

Rituximab

A case report of the successful use of the anti-CD20 humanized mouse antibody rituximab has provided preliminary evidence for its potential effectiveness in treating aplastic anemia.²²¹ Clinical trials have not examined its efficacy compared to standard immunotherapy (ATG and cyclosporine), in patients refractory to standard therapy, or as a third drug in an immunotherapy regimen. Whether B lymphocytes play a role in the pathogenesis of T-cell–mediated aplastic anemia has not been defined, so rituximab does not appear to have a theoretical rationale for use at this time. However, a singular case of antibody-mediated aplastic anemia responded to rituximab, and the autoantibodies became undetectable.^222,223

Other Non-immune Therapies

Androgens

Randomized trials have not shown efficacy when androgens were used as primary therapy for severe aplastic anemia.^224,225 Androgens stimulate the production of erythropoietin, and their metabolites stimulate erythropoiesis when added to marrow cultures in vitro. High doses of androgens were beneficial in some patients with moderately severe aplasia.²²⁴ Series of patients were reported in which survival seemed improved as compared with historical controls, but this could have resulted from improved supportive care.¹⁴⁴ Masculinization and other androgen side effects can be severe, especially in female patients. Long-term survivors after androgen therapy have essentially the same progression to clonal hematologic disorders as patients treated with immunosuppressive agents.¹⁴⁴ These agents have been replaced by immunosuppression or allogeneic hematopoietic stem cell transplantation as a principal approach to treatment.

Cytokines

Granulocyte Colony-Stimulating Factor

Despite their effectiveness in accelerating recovery from chemotherapy, these agents have been far less effective in achieving long-term benefits in patients with severe aplastic anemia. Daily treatment with G-CSF²²⁶ has improved marrow cellularity and increased neutrophil counts approximately 1.5- to 10-fold. Unfortunately, in nearly all patients, the blood counts return to baseline within several days of cessation of therapy. Although occasional patients show evidence of trilineage marrow recovery with long-term therapy, the vast majority do not respond. Therapy with myeloid growth factors is probably best reserved for episodes of severe infection or as a preventive measure prior to dental work or other procedures that would compromise mucosal barriers in patients who have not responded to stem cell transplant or immunotherapy. G-CSF in a dose of 5 mcg/kg by subcutaneous injection is easiest to administer and seems to be associated with the fewest side effects. The drug can be given daily or fewer times per week depending on the response. Newer pegylated preparations have a longer effect and usually are administered at less frequent, every-other-week intervals. The SAA Working Party of the European Group for Blood and Marrow Transplant reported that G-CSF added to ATG and cyclosporine reduces infection early in treatment, but does not affect survival or length of remission.²⁰³ Generally, prophylactic use of growth factors is not warranted.

Interleukin-1

IL-1, a potent stimulator of marrow stromal cell production of other cytokines, and IL-3 have been ineffective in small numbers of patients so treated with severe aplastic anemia.^227,228 These disappointing results with cytokines are not unexpected, as previous work has found high serum levels of growth factors in patients with aplastic anemia. Moreover, the majority of patients have suppression of very primitive progenitors, which may be unresponsive to individual factors that act on more mature progenitor cells.

Eltrombopag

Eltrombopag is a thrombopoietin (TPO) receptor agonist used to stimulate platelet production in patients with immune thrombocytopenia. It is a relatively small molecule of the biaryl hydroxone class,^228a is well absorbed from the gastrointestinal tract, and is thus administered orally. It binds to the transmembrane portion of the thrombopoietin receptor (cMpl) and induces signal transduction through the mitogen-activated protein kinase (MAP) and Janus kinase (JAK) pathways. TPO may expand stem cell numbers and promote DNA repair.^229,230 In addition to its use in ITP, where it can induce megakaryocyte maturation and enhance platelet production, trials of eltrombopag in severe aplastic anemia have found that a multilineage hematopoietic response occurred leading to restoration of granulopoiesis, erythropoiesis, and megakaryocytopoiesis with a subsequent increase in neutrophil, erythrocyte, and platelet counts, occasionally to normal levels.^231,232,213a MPL receptors are present on hematopoietic stem cells, and eltrombopag interacts with marrow stem cells through these receptors, inducing a multilineage progenitor cell proliferative and maturation effect.

Use of eltrombopag as a single agent in 43 patients with acquired aplastic anemia who did not respond to immunotherapy resulted in improved hematopoiesis and cell counts in 17 (40 percent), with several having improved bi- or trilineage hematopoiesis and cell counts. Five patients had near normalization of all blood counts and had therapy stopped after 9 to 37 months with maintenance of their blood counts for 1 to 13 months of observation.²³³ Although many did not normalize their counts, several became red cell and platelet transfusion independent. Eight patients developed new cytogenetic abnormalities (5 of 8 patients developed −7 or del[7]), but none progressed to AML. In subsequent studies of patients with aplastic anemia, the response rates to traditional immunosuppression were improved significantly when eltrombopag was added to the standard two-drug (ATG and cyclosporine) immunotherapy protocol.^213a (See “Combination of Immunotherapy and Eltrombopag,” above.) The FDA approved the use of eltrombopag for patients with severe aplastic anemia in 2014.

Splenectomy

Removal of the spleen does not increase hematopoiesis but may increase neutrophil and platelet counts two- to threefold and improve survival of transfused red cells or platelets in highly sensitized individuals.²³⁴ The surgical morbidity and mortality in patients with few platelets and white cells makes this a questionable therapeutic procedure. Because there are more successful methods of therapy that attack the fundamental problem, this approach is not recommended.

Other Therapy

High doses of intravenous γ-globulin have been given to small numbers of patients with severe aplastic anemia^235,236 because of its success in treating certain cases of antibody-mediated pure red cell aplasia. Some improvement was noted in 4 of 6 patients treated. Another treatment that is occasionally successful is lymphocytapheresis to deplete T cells.^237,238 Agents that target other T-cell functions, such as alefacept, a CD2-directed leukocyte function antigen-3 (LFA-3)/Fc fusion protein that consists of the extracellular CD2-binding portion of the human LFA-3 linked to the Fc (hinge, CH2 and CH3 domains) portion of human immunoglobulin (Ig) G_l are being tested as immunosuppressive drugs in acquired aplastic anemia.²³⁹

Course and Prognosis

At diagnosis, the prognosis is largely related to the absolute neutrophil and platelet count. The absolute neutrophil count is the most important prognostic feature, with a count of fewer than 500/μL (0.5 × 10⁹/L) considered severe aplastic anemia and a count of fewer than 200/μL (0.2 × 10⁹/L) very severe aplastic anemia, the latter associated with a poor response to immunotherapy and usually a dire prognosis, if early successful allogeneic transplant is not available. In the past, the prognosis appeared worse when the disease followed hepatitis.^74,75 But, more comprehensive results with immunosuppression²¹⁴ or hematopoietic stem cell transplantation²⁴⁰ show an equivalent response to that seen with idiopathic or drug-induced cases.²⁴¹

Before marrow transplantation and immunosuppressive therapy, more than 25 percent of the patients with severe aplastic anemia died within 4 months of diagnosis; half succumbed within 1 year.^238,242 Marrow transplantation is curative for approximately 80 to 90 percent of patients younger than 20 years of age, approximately 70 percent if between the ages of 20 and 40 years, and approximately 50 percent if older than age 40 years.^160,243 Unfortunately, as many as 40 percent of transplant survivors suffer the deleterious consequences of chronic graft-versus-host disease,¹⁶⁰ and the risk of subsequent cancer can be as high as 10 percent in older patients or after immunotherapy prior to hematopoietic stem cell transplantation.^244,245 The best outcomes occur in those patients who have an allele-based HLA-matched sibling; have not been exposed to immunosuppressive therapy prior to transplantation; have not been exposed and sensitized to blood cell products; have had a marrow rather than a blood stem cell donor product; and have not been subjected to high-dose radiation in the conditioning regimen for transplantation.^{160,244,246,247}

Combination immunosuppressive therapy with ATG and cyclosporine leads to a marked improvement in approximately 70 percent of the patients; a higher initial absolute reticulocyte and lymphocyte counts are predictive of the response to therapy.²⁴⁸ A high proportion of Treg B cells is associated with an increased likelihood of a favorable response to immunotherapy.⁴⁰ Treg B lymphocytes can be expanded in vitro with interferon. Addition of low-dose IL-2 to current regimens may improve the results of immunotherapy. This suggestion awaits the results of clinical trials.⁴⁰ Although some patients regain normal blood counts, many continue with moderate anemia or thrombocytopenia. In as many as 40 percent of patients initially responding to immunosuppressive therapy, their disease may relapse or progress to PNH, a myelodysplastic syndrome, or AML over 10 years of observation. Moreover, the beneficial effects of immunotherapy are often lost 10 years after treatment. In 168 transplanted patients the actuarial survival at 15 years was 69 percent, and in 227 patients receiving immunosuppressive therapy it was 38 percent.¹⁸¹ The long-term survival in pediatric patients younger than age 18 years appears better, with approximately one-third relapsing at 10 years.²⁴⁹

Treatment with high-dose cyclophosphamide produces early results similar to that seen with the combination of ATG and cyclosporine.^250,251 However, cyclophosphamide has greater early toxicity and slower hematologic recovery, but may generate more durable remissions. Its use has been too limited to reach a firm conclusion on its relative merits and it is rarely used as the first choice of immunotherapy.

FANCONI ANEMIA

Definition and History

Fanconi anemia is the most common form of constitutional aplastic anemia and was initially described in three brothers by Fanconi in 1927.²⁵² It is inherited as an autosomal recessive condition that results from defects in genes that modulate the stability of DNA.

Epidemiology

Fanconi anemia is an uncommon disorder and is estimated to be present in 1 in 1 million individuals. It is far more frequent in Afrikaners of European descent.²⁵³ This unusually high frequency has been attributed to a founder effect.

Etiology and Pathogenesis

Sixteen complementation groups, defined by somatic cell hybridization, are associated with the development of Fanconi anemia (FA).^254,255 A complementation group is a genetic subgroup. Identifying a complementation group requires adding a gene to the genome of a cell to correct (complement) the genetic defect. This procedure can be done by cell fusion studies. After fusing two cells together, thereby joining their genetic material, one can test the cells for the genetic defect. In the case of Fanconi anemia, this would be with the diepoxybutane test. In this test, diepoxybutane results in chromosome fragmentation in the cells of patients with Fanconi anemia. Hybrids in which the hypersensitivity to diepoxybutane is corrected (complemented) can be assumed to result from the fusion of cells from different genetic subgroups (complementation groups), whereas hybrids that still show the sensitivity are the result of fusion of cells from the same subgroup. Because one can determine the complementation group without knowing the gene involved, this approach is the first step in understanding the genetic basis of a disease. Once the genes are known, one does not need to use cell fusion studies; rather, retroviral vectors can be used to insert corrected genes into the cells.

The complementation groups have been designated FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, and Q. Table 35–8 lists the gene mutations corresponding to these complementation groups, and Fig. 35-5 summarizes the functions of the known Fanconi anemia proteins.²⁵⁵ The great majority of patients have mutations of FANCA, FANCC, or FANCG.²⁵⁶ It has been proposed that the A and C gene products, which are cytoplasmic proteins, form an “FA core complex” with the products of genes B, E, F, G, L, and M, which are adaptors or phosphorylators.^256,257 The complex translocates to the nucleus, where it is required for the ubiquitination of FANCD2 and protects the cell from DNA crosslinking and participates in DNA repair (Fig. 35-6). DNA damage initiates activation of the FA/BRCA pathway and ubiquitination of FANCD2, which is targeted to the altered DNA and facilitates repair by interacting with DNA repair proteins, BRCA1, FANCD1/BRCA2, FANCN/PALB2, and RAD51. In the presence of a mutant gene product, the normal protective and repair functions are disturbed leading to damaging effects in sensitive tissues, including hematopoietic cells. The genetic damage appears related to the adverse effects of reactive oxygen radicals, such as superoxide and hydrogen peroxide as well as aldehydes produced by normal cellular metabolism.^258-260 In addition to the genetic defects leading to DNA instability and an inability to repair DNA, TNF-α and -γ are overexpressed in the marrow of Fanconi anemia patients.²⁶¹ The excess TNF-α may play a role in the suppression of erythropoiesis in these patients.

The generation of reactive oxygen radicals and aldehydes, the defective mechanisms of DNA repair, the hypersensitivity to cytokines such as TNF-α, and the age-related shortening of DNA-protective telomeres produce a marked predisposition to clonal evolution and neoplasia in Fanconi anemia patients (see “Therapy and Course” below).

Clinical Features

Growth retardation, resulting in short stature, and skeletal anomalies are common. Absent, misshapen, or supernumerary thumbs and dysplastic radii occur in half the patients. Hip and vertebral abnormalities also may occur. Septal heart defects, eye abnormalities, and absent, misshapen, or fused kidneys may be present. Females may have aplasia of the uterus and vagina, absent ovaries, infertility and late menarche and early menopause and males may have hypospermia. Thus, hypogonadism may be evident. Learning disability is frequent, and microcephaly and mental retardation may be a feature. The skin may be generally hyperpigmented or may have areas of abnormal skin pigmentation referred to as café-au-lait spots, which are flat, light brown, and from 1 to 12 centimeters in diameter. Hepatosplenomegaly is not a feature of the disease. Some patients have no or minor phenotypic abnormalities and may be diagnosed as a result of the onset of marrow failure or a cancer involving any of many sites as late as the fifth decade of life.

The onset of marrow failure is gradual and usually is evident during the last half of the first decade of life. The manifestations of anemia, including weakness, fatigue, and dyspnea on exertion, and of thrombocytopenia with epistaxis, purpura, or other unexpected bleeding, are the principal findings. Hematologic and visceral manifestations are combined eventually in more than a third of patients, but some may have cytopenias and inconspicuous somatic changes, whereas others may have somatic anomalies with no or a nominal disorder of blood cell formation for months or years. Some who carry the gene may be virtually unaffected.^262-264 In a review of the more than 1300 patients in the literature, 100 patients, or fewer than 7 percent, without anomalies were identified by chromosome breakage studies (see “Laboratory Features” below) because of affected siblings.²³⁰ In the past, children in Fanconi families with an onset of aplastic anemia without congenital somatic abnormalities were thought to have a different disorder termed Estren-Dameshek syndrome.²⁶⁵ However, these children, whose lymphocytes show sensitivity to diepoxybutane, are considered to have Fanconi anemia without skeletal abnormalities.

Laboratory Features

Blood counts and marrow cellularity are often normal until 5 to 10 years of age, when pancytopenia develops over an extended interval. Macrocytosis with anisocytosis and poikilocytosis may be present before any cytopenia occurs. Thrombocytopenia may precede the development of granulocytopenia and anemia. The marrow becomes hypocellular, and in vitro colony assays reveal a decrease in CFU-GM and BFU-E.²⁶⁴

Random chromatid breaks are present in myeloid cells, lymphocytes, and chorionic villus biopsy samples. This chromosome damage is intensified after exposure to DNA crosslinking agents such as mitomycin C or diepoxybutane. The hypersensitivity of the chromosomes of marrow cells or lymphocytes to the latter agent is used as a diagnostic test for this condition. Cell-cycle progression is prolonged at the G2-to-M transition, and the cells are more susceptible to oxygen toxicity when cultured in vitro. It is important to test the lymphocytes from pediatric patients with aplastic anemia for sensitivity to diepoxybutane, because therapy for Fanconi anemia differs from that used for acquired aplastic anemia.

In the near future, clinical laboratories will be able to genotype suspected patients. Determining the specific gene mutation responsible in a patient (see Table 35–8) is important because it confirms the diagnosis, identifies the genotype linked to BRCA2 that may predispose to a cancer (e.g., breast, ovary), and permits carrier detection.²⁶⁶

Differential Diagnosis

The differential diagnosis of Fanconi anemia includes other causes of aplastic anemia, particularly those familial syndromes associated with skeletal anomalies and other dysmorphic features. Other familial types of aplastic anemia have been reported with or without associated anomalies. In those instances in which no sensitivity to DNA damaging agents is observed, the syndrome does not represent Fanconi anemia. Several uncommon syndromes of this type are described below and are tabulated in Table 35–9.

Therapy and Course

Most patients with Fanconi anemia do not respond to ATG or cyclosporine but do improve with androgen preparations, often for as long as several years. Cytokines may provide some improvement in blood counts, but their effect may wane. Studies in a mouse model also suggest that cytokine effects may not be sustained.²⁸⁷ The cumulative median survival is approximately 20 years from progressive marrow failure, conversion to myelodysplastic syndrome, AML (approximately 10 percent of patients), or the development of a variety of other cancers, such as those involving the genitourinary system, digestive system (especially liver), or head and neck.²⁸⁸ Multiple cancers in an individual patient also occur. Cancers may occur as late as the fifth decade of life and precede the diagnosis of Fanconi anemia in 25 percent of patients.²⁸⁸ The presence of a clonal cytogenetic abnormality or marrow morphology consistent with myelodysplasia markedly reduces the 5-year survival.²³⁵ Allogeneic hematopoietic stem cell transplantation is curative for the marrow manifestations of Fanconi anemia.^289-292 A marked reduction in dosage of the marrow-conditioning regimen of cyclophosphamide and radiation is necessary owing to the undue sensitivity of the tissues to DNA-damaging exposures. The risk of cancer is so high that, where practical, surveillance should be used; for example, frequent pelvic exams in females, hepatic ultrasonography to detect adenomas, and careful oropharyngeal examinations. Therapy of cancer in patients with Fanconi anemia needs to consider the marked sensitivity of their cells to DNA cross-linking agents and radiotherapy.

Normal complementary DNA has been transferred into cells from patients with restoration of resistance to DNA damaging agents.^293,294 Difficulties in this approach include the paucity of stem cells in these patients, as well as the potential toxicity of the gene transfer methodology.

DYSKERATOSIS CONGENITA

Definition

This inherited disorder is characterized by cutaneous and mucous membrane abnormalities, progressive marrow insufficiency, and a predisposition to malignant transformation. It is much more common in males than in females, and occurs in approximately 1 per 1,000,000 population.^254,295

Pathogenesis

Dyskeratosis usually is inherited as a recessive X-chromosome–linked disorder although rare cases can have autosomal dominant or autosomal recessive inheritance (Table 35–10). The disease is a reflection of telomere complex dysfunction,^296-299 and it results from defective telomerase activity resulting from mutations in the telomerase-related genes (Fig. 35-7).^297,300,301 The telomerase complex maintains the length of telomeres, which are nucleotide tandem repeat structures residing at the termini of eukaryotic chromosomes (e.g., 5′-TTAGGG-3′). Telomerase restores the guanine (G)-rich telomere repeats that are lost as a result of end-processing during normal cell division. Combined with protein, located at the ends of chromosomes, they maintain chromosome integrity by preventing end-to-end chromosome fusion, preventing chromosome degradation, and preventing chromosome instability. In dyskeratosis congenita, the telomeres are markedly shortened resulting in genomic instability and cell (including marrow cell) apoptosis, and the underlying gene defects may alter Box H/ACA small nucleolar RNAs, such as the telomerase RNA component, TERC, that is central to telomere maintenance.³⁰² Rapidly proliferating cells are at highest risk for dysfunction. Mutations of the DKC1 gene are responsible for the X-linked recessive form. DKC1 encodes dyskerin, which is a conserved multifunctional protein component of the telomerase complex. Mutations of the TERT, TERC, and TINF2 genes are the principal abnormalities in the autosomal dominant form. TERC is the RNA component of the telomerase reverse transcriptase that TERT, the reverse transcriptase, uses to synthesize the 6-bp repeats on the 3′ end of telomeric DNA. Mutations of TINF2 have been described in patients with dyskeratosis congenital.³⁰³ TINF2 is a component of the shelterin complex, which prevents end-to-end telomere fusion.³⁰³ It also permits the distinction of telomeres from sites of DNA damage, preventing their inappropriate processing. Recessive mutations in NHP2 and in NOP10, which encode parts of small ribonucleoprotein components associated with the telomerase complex, also have been described in association with dyskeratosis.^304,305 Homozygous recessive mutations in the TERT gene produce a severe variant of dyskeratosis, referred to as the Hoyeraal-Hreidarsson syndrome.²⁹⁵

Clinical Findings

The cutaneous findings usually appear after 5 years of age and include reticulated, tan to gray, hyperpigmented and hypopigmented cutaneous macules; alopecia of scalp, eyelashes, and eyebrows; adermatoglyphia (loss of dermal ridges on fingers and toes); hyperkeratosis of palms and soles; mucosal leukoplakia in 75 percent of patients; and dystrophic nails in more than 85 percent of patients.^254,295,296 Other mucosal sites, such as conjunctiva, lacrimal duct, esophagus, urethra, vagina, and anus, can be involved, sometimes with stenosis leading to dysphagia or dysuria. Pulmonary vascular involvement occurs in a significant minority of affected children. Aplastic anemia usually develops in late childhood or early adulthood and is evident in the classical blood and marrow findings described under acquired aplastic anemia. Female carriers of X-linked dyskeratosis congenital may have slight abnormalities such as a dystrophic nail, a single area of hypopigmentation, or slight leukoplakia.²⁹⁵ The clinical manifestations exhibit disease anticipation, occurring earlier in subsequent generations, and this appears related to earlier shortening of the telomeres.³⁰⁷

Diagnosis

The diagnosis results from the combination of phenotypic findings and blood cell deficiencies. Genetic analysis for telomerase complex gene mutations should be used to confirm the clinical conclusion. Shortened telomere length in leukocytes also can be assessed by flow cytometric fluorescence in situ hybridization studies.³⁰⁸

Management

Stem cell hematopoietic transplantation has had inconsistent results because of frequent and severe posttransplantation complications.³⁰⁹ Nonmyeloablative transplantation might improve results.^310-312 Transplantation might improve the cytopenias, but not the abnormalities of other organs or the frequency of secondary nonhematopoietic cancer.

Course and Prognosis

The incidence of squamous cell carcinoma of mucosal sites is increased and the squamous cell carcinoma often originates in sites of leukoplakia in the skin, gastrointestinal, or genitourinary tracts.³¹³ These carcinomas usually develop between the ages of 20 and 30 years. Mortality from neutropenic infection or thrombocytopenic hemorrhage occurs in about two-thirds of patients with aplastic anemia. Median survival is approximately 30 years.

SHWACHMAN-DIAMOND SYNDROME

Definition

This disease is an uncommon inherited disorder that is estimated to occur once in every 75,000 births,³¹⁴ manifesting exocrine pancreatic insufficiency with secondary steatorrhea, blood cell deficiencies, and skeletal abnormalities. It was first described in 1964.^315,316

Pathogenesis

Shwachman-Diamond syndrome results from mutations in the SBDS gene on chromosome 7q11, which induces accelerated cellular apoptosis via the FAS pathway.³¹⁷ The resulting hyperproliferation may account for the abnormal telomere shortening that has been documented in the leukocytes in this condition.³¹⁸ The pathogenetic mechanism that (1) prevents development of pancreatic acinar cells, (2) results in abnormal bone morphogenesis, and (3) causes marrow impairment of blood cell production is not understood. SBDS knockdown in experimental animals affects expression of genes involved in brain, bone, and marrow development, and may be the result of the gene’s role in RNA processing.^319,320 The SBDS gene promotes the release of eukaryotic initiation factor 6 from the pre-60s ribosome.³⁰² This action is necessary for the formation of a mature 80s functional ribosome and production of appropriate ribosome joining. The mutations in SBDS also result in abnormalities in neutrophil motility and chemotaxis, but pus formation in vivo seems adequate.

Clinical Findings

Pancreatic insufficiency, steatorrhea, and neutropenia are present in most patients at the time of diagnosis.^315,316,321 Pallor may reflect anemia and easy bruising; epistaxis or bleeding from other sites reflect thrombocytopenia. Neutropenia occurs in approximately 95 percent, anemia in approximately 50 percent, and thrombocytopenia in approximately 35 percent of patients.³¹⁷ Thus, a substantial plurality of patients has bicytopenia or tricytopenia with an hypoplastic marrow. Fetal hemoglobin levels are elevated in approximately 75 percent of the patients, perhaps secondary to erythroid hypoplasia. Cytogenetic abnormalities involving chromosomes 7 and 20 have been described in marrow cells. Nutritional inadequacies related to intestinal malabsorption result in a failure to thrive. Short stature is characteristic. Skeletal abnormalities are present in most patients, notably osteopenia, but also syndactyly, supernumerary metatarsals, coax vera deformity, and dental enamel defects and caries. Hepatic dysfunction as evidenced by elevated serum aminotransaminase is seen in most young patients and appears to resolve with age.³²² Delayed puberty is common. The neutropenia and chemotactic abnormality may result in recurrent infections, including sinusitis, otitis, pneumonia, osteomyelitis, and others. Pancreatic cell lipase production improves with age, and as many as half the patients may have improvement in lipid absorption in the small bowel with time.

Diagnosis

The diagnosis is based on the clinical findings of failure to thrive, steatorrhea, and neutropenia. Pancreatic insufficiency can be established by low serum trypsinogen in patients younger than 3 years of age. The marrow may be initially normal, but develops evidence of marrow failure and sometimes cytogenetic abnormalities, particularly of chromosome 7, as the child ages. The age of expression of clonal hematopoiesis (e.g., myelodysplastic syndrome or AML) is variable.³²³ The SBDS gene mutation is present in 90 percent of patients with Shwachman-Diamond syndrome.³²⁴ The remaining 10 percent have the clinical features of the syndrome, but the gene defects have not been defined.

Management

Supportive care, particularly with supplemental pancreatic enzymes, to provide proper nutrition, and appropriate and prompt treatment of bacterial infections with antibiotics is important. Many agents, including G-CSF, glucocorticoids, pancreatic extract, vitamins, have been tried to improve the neutropenia with unpredictable results. Some agents have potential risks, such as G-CSF fostering clonal evolution and glucocorticoids fostering immunodeficiency. Severe hematopoietic dysfunction and cytopenias can be corrected with allogeneic hematopoietic stem cell transplantation.³²⁵

Course and Prognosis

Death from overwhelming sepsis is common. These patients, especially males, have a significant risk of progression to a myelodysplastic syndrome or AML.^316,326,327 Cytogenetic abnormalities are common, and telomeres are shortened,³²⁸ as in other marrow failure syndromes. Survival is a function of the severity of the cytopenias. If the cytopenias are mild, survival is not uncommon into the fourth or fifth decade of life. It is not clear whether Shwachman-Diamond syndrome is associated with an increased incidence of solid tumors.

OTHER INHERITED APLASTIC ANEMIAS

Several other rare syndromes are associated with aplastic pancytopenia, and these are described in Table 35–9. Congenital (hereditary) amegakaryocytic thrombocytopenia (CAMT) results from mutations in the TPO receptor gene, MPL.^270-272 The affected children can be divided into two groups: CAMT I with mutations leading to complete loss of function of the TPO receptor, resulting in more severe thrombocytopenia and a rapid progression to pancytopenia (aplastic anemia), and CAMT II, resulting from a variety of missense mutations, in which affected children have an increase in platelet count above 50 × 10⁹/L with time and much slower progression to and sometimes less-severe pancytopenia.²⁷¹ Reticular dysgenesis results from a pluripotential stem cell defect as both lymphoid and granulocytic progenitors are affected.^280,281 It is a rare autosomal recessive disorder caused by mutations in the adenylate kinase 2 gene (AK2) and characterized by bilateral sensorineural deafness, severe combined immunodeficiency, and agranulocytosis, which subjects infants to severe, often, life-threatening infections. The Seckel syndrome results from mutations in the ATR gene, and marrow cells exhibit heightened sister chromatid exchange. The ataxia-telangiectasia mutated and rad3-related (ATR) kinase mediates cellular responses to DNA damage and replication stress. Most of the eight syndromes delineated in Table 35–9 can be treated by marrow transplantation, but this step, if successful, does not correct somatic abnormalities, only the hematopoietic and immunologic defect. The restoration of robust lymphohematopoiesis by transplantation may decrease their propensity to undergo clonal evolution to a clonal myeloid or, in some cases, lymphoid disorder.