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Aplastic Anemia
Aplastic anemia (AA), the paradigm of the bone marrow (BM) failure syndromes, is most simply defined as peripheral blood pancytopenia and a hypocellular BM ( Fig. 31.1 ). AA occurring as a primary hematologic disorder is historically denoted idiopathic, but it is now understood as immune-mediated. Constitutional marrow failure syndromes share a similar pathology of an apparently “empty” marrow (see Chapter 30 ) and can be distinguished from immune AA by personal and family history, age of onset, involvement of other organs, and genomic testing. Physical and chemical damage (most frequently as radiation and chemotherapy) also can lead to an aplastic marrow. However, the diagnosis of AA requires excluding other causes of pancytopenia, especially hypocellular myelodysplasia (see Chapter 61 ). Even typical AA can vary in its clinical course, from a fulminant illness marked by hemorrhage and infections to an indolent process manageable by transfusions. Immune AA is treated by hematopoietic stem cell transplantation and also immunosuppressive therapies; both result in excellent outcomes. The reader is referred to previous editions of this textbook for references, as well as to the authors’ recent reviews.
BONE MARROW MORPHOLOGY IN SEVERE APLASTIC ANEMIA.
Bone marrow biopsy specimen, of sufficient length (A) shows severe hypocellularity. The corresponding aspirate (B, C, D) shows empty marrow spicules and residual stoma including lymphoid cells, plasma cells, histiocytes and mast cells.
History
The study of BM failure dates to 1888, when Paul Ehrlich described a young woman who died after an explosive short illness, marked by severe anemia, bleeding, and high fever. As a pathologist, Ehrlich was struck by the absence of nucleated RBCs and the fatty quality of the femoral BM. Vaquez and Aubertin, in a 1904 case report of “pernicious anemia with yellow BM,” named the disease and emphasized a pathophysiology of anhematopoiesi s. The etymologic root of the term aplastique is the Greek verb πλάϑω , to create and give shape to ( άπλαζτκή , the adjective, unformed).
Classification
Historically, AA has been classified by clinical associations ( Table 31.1 ). A hypocellular marrow is typical following irradiation and exposure to cytotoxic chemotherapy. AA has been associated with the use of chemicals and drugs, viral infections, and with other diseases ( Table 31.2 ). The same pathology occurs in constitutional syndromes, often as a component of multiorgan involvement (see Chapter 30 ). Historical associations of environmental exposures and causation are interesting but should be considered with some skepticism because of biases of observation and reporting, and lack of direct evidence. Most patients with sporadic disease have been considered “idiopathic” but their AA can now be considered immune-mediated. A simple classification of AA divides the cases into three categories: (1) marrow damage from toxicity, which is usually dose-dependent and reversible; (2) constitutional, as part of a recognizable gene defect and due to an identifiable genetic aberration; and (3) immune AA.
Table 31.1
Differential Diagnosis of Pancytopenia
| Pancytopenia With Hypocellular Bone Marrow |
|
| Pancytopenia With Cellular Bone Marrow |
|
| Hypocellular Bone Marrow ± Cytopenia |
|
a Pancytopenia in tuberculosis only rarely is associated with a hypocellular bone marrow at biopsy or autopsy. Marrow failure in the setting of tuberculosis is almost always fatal; exceptional patients probably had underlying myelodysplasia or acute leukemia.
Table 31.2
A Classification of Aplastic Anemia
| Acquired Aplastic Anemia |
|
| Constitutional Aplastic Anemia |
|
Epidemiology
Incidence, Geographic and Age Distribution
The International AA and Agranulocytosis Study (IAAAS) was conducted in Europe and Israel from 1980 to 1984. This study was performed prospectively and applied strict case definition to pathologically confirmed cases. Using stringent criteria, the overall annual incidence of AA was 2 cases per 1 million people. In Asia, similar methodology was applied by Thai investigators to determine a higher annual incidence, 4.0 cases per 1 million people in Bangkok, and 5.6 cases per 1 million people in the northeastern Thai province of Khonkaen. In general, from published, hospital-based series, personal communications, and first-hand observations, AA appears more prevalent in less developed regions of the world. There are no major sex or racial differences in the occurrence of AA. AA is a disease of the young ( Fig. 31.2 ). Most patients present between 15 and 25 years of age or older than 60 years of age.
Epidemiologic Clues to Causality
Population-based studies have investigated possible causal associations. Drugs are implicated in approximately 25% of the cases of AA in the West; while in Thailand, AA was attributed to drug exposure in only approximately 15% of the cases. There are associations with chemical exposures, exposures to viruses, hepatitis, and occupation. There is evidence that geographic variation in the incidence of AA might result from environmental causes and also genetic predispositions.
Genetic Aspects
In children and young adults, acquired AA should be distinguished from the main inherited forms of BM failure, Fanconi anemia (FA) and telomere biology disease (TBD) (see Chapter 30 ). Identification of constitutional AA has important therapeutic implications. Patients with FA and TBD can lack typical physical anomalies, and the pancytopenia can develop long after childhood, mimicking acquired disease. Genomic approaches to the study of AA are likely to uncover other genetic contributions to susceptibility to BM failure.
A few histocompatibility types have also been associated with AA, most consistently human leukocyte antigen (HLA)-DR2. HLA-DR subtypes predicted response to immunosuppressive therapy in a large cohort of US AA patients, in which HLA-DR15 was associated with the presence of a paroxysmal nocturnal hemoglobinuria (PNH) clone (see Chapter 32 ) and responsiveness to immunosuppression. Genetic predisposition may be responsible for some idiosyncratic reactions to drugs and chemicals leading to the development of AA. Polymorphisms in cytokine genes, associated with an increased immune response, are also more prevalent in AA. Genome-wide transcriptional analysis of T and natural killer cells from AA patients has implicated pathologic expression of components of innate immunity, including Toll-like receptors.
Etiology And Pathogenesis
Hematopoiesis in Bone Marrow Failure
Stem Cells
A consistent laboratory finding for patients with AA is a very low number of hematopoietic progenitor cells. Deficient in vitro colony formation by BM cells from AA patients is observed, even in the presence of high levels of hematopoietic growth factors. The total number of progenitors in a BM sample is reduced, and the number of progenitor cells assayed from a purified CD34 + cell population is low. The numbers of long-term culture-initiating cells (LTC-ICs), and in vitro surrogate stem-cell assays (see Chapter 9 ) are also profoundly deficient in patients with severe AA. At clinical presentation, the number of LTC-ICs is usually less than 10% of normal; combined with a reduction in total BM cellularity to 10% or less, the stem-cell number is estimated to be reduced to 1% or less than normal in patients with AA ( Fig. 31.3 ).
Telomeres and Bone Marrow Failure
One peculiar feature of white blood cells in some cases of AA is short telomeres. The discovery by linkage analysis in large pedigrees that the X-linked form of dyskeratosis congenita (DKC) was caused by mutations in DKC1 and subsequently purposeful identification of mutations in TERC in some autosomal dominant patients with this constitutional BM failure syndrome provided a genetic basis for DKC (see Chapter 30 ). Central to the repair machinery is an RNA template, encoded by TERC , on which telomerase, a reverse transcriptase encoded by TERT , elongates the nucleotide repeat structure; other proteins, including the DKC1 gene product dyskerin, are associated with the telomere repair complex. Systematic surveys of DNA disclosed first telomerase RNA component ( TERC ) and later telomerase reverse transcriptase ( TERT ) mutations in some patients with apparently acquired AA, including older adults. Family members who share the mutation, despite normal or near-normal blood counts, have hypocellular marrows, reduced CD34 + cell counts and poor hematopoietic colony formation, increased hematopoietic growth factor levels, and of course short telomeres. However, clinical presentation is much later than in typical DKC, and physical anomalies are often absent. Chromosomes are also protected by shelterin proteins that bind directly to telomeres. Mutations in the shelterin gene lead to very severe DKC. Some inherited sequence variants/polymorphism in genes that repair or protect telomeres may be genetic risk factors in acquired AA, probably because they confer a quantitatively reduced hematopoietic stem cell compartment that may also be qualitatively inadequate to sustain immune-mediated damage. Accelerated telomere attrition in AA not currently explained by mutations may be caused by subtle or obscure genetic lesions, or follow BM stress and excessive stem cell turnover.
Stromal and Hematopoietic Growth Factors
Stromal cell function is usually not defective in cases of AA. Adherent cells from patients support hematopoiesis by normal CD34 + cells, whereas no hematopoietic colonies develop when AA patient CD34 + cells are cultured in the presence of normal stroma ( Fig. 31.4 ). Stromal cells cultured from patient’s BM generally produce normal quantities of hematopoietic growth factors. Serum levels of erythropoietin, thrombopoietin, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are almost always normal or elevated. Adequate stromal function is implicit in the success of BM transplantation in AA because important stromal elements remain of host origin.
Pathophysiologic Pathways Leading To Aplastic Anemia
Direct Hematopoietic Injury
The most common cause of BM hypoplasia is iatrogenic; transient BM failure routinely follows treatment with cytotoxic chemotherapeutic drugs or irradiation. Certain chemical or physical agents directly injure proliferating and quiescent hematopoietic cells. However, patients with community-acquired AA rarely have a history of exposure to such physicochemical agents. Even benzene, which can act as a particularly inefficient cytotoxic chemical, is an infrequent cause of AA in developed countries. Drugs are associated with acquired AA, and in some instances, they can directly cause BM damage. Compared with chemotherapeutic agents, which are delivered in high doses, relatively low total quantities of ingested drug apparently cause idiosyncratic hematologic reactions. In addition to their direct toxic effects, chemicals and viruses may induce complex and not well-understood immune reactions leading to BM failure.
Immune-Mediated Bone Marrow Failure
In the 1970s, Mathé and colleagues observed an unexpected improvement of pancytopenia due to AA after failed BM transplantation. They speculated that the immunosuppressive conditioning regimen, intended to allow engraftment of the donor BM, might instead have promoted the recovery of host BM function. The effectiveness of diverse treatments that reduce lymphocyte number or block T-cell function, and the superior results obtained when agents are combined strongly suggest that such therapeutic success is caused by the immunosuppressive effects of the drugs used. AA shares clinical and pathophysiologic features with other autoimmune or immune-mediated human diseases that are also characterized by T-cell–mediated, tissue-specific organ destruction (inflammatory bowel disease, type 1 diabetes, multiple sclerosis, uveitis, and others).
Immune system destruction of BM occurs in animal models of graft-versus-host disease (GVHD) and in humans with transfusion-associated GVHD, in which AA is the cause of death. Very small numbers of effector cells, which have been conveyed by residual lymphocytes contained within the transfusion product or with solid organ transplants, are sufficient to mediate GVHD under these conditions. AA is associated with rheumatologic syndromes, such as eosinophilic fasciitis, and with systemic lupus erythematosus. AA occasionally occurs in individuals with hypogammaglobulinemia or congenital immunodeficiency syndrome, thymoma, thymic hyperplasia, and thymic carcinoma. AA is a rare complication of immunotherapy of cancers, both checkpoint inhibitors and chimeric antigens receptor T cells (CAR-T cells).
Laboratory support for the immune hypothesis first came from coculture experiments in which mononuclear cells from AA patient’s blood or BM were shown to suppress in vitro colony formation by hematopoietic progenitor cells. T-cell depletion sometimes improved colony formation in vitro. Patient’s blood and BM cells were shown to produce a soluble factor that inhibited hematopoiesis, ultimately identified as interferon (IFN)-γ. Patient’s cells overproduce IFN-γ and tumor necrosis factor (TNF), two cytokines that inhibit hematopoietic proliferation. The T-box transcription factor, Tbet, which is critical to Th1 polarization, is constitutionally expressed in a majority of AA patients. AA blood and BM also contain elevated numbers of activated cytotoxic lymphocytes, and activity and levels of these cytotoxic cells are decreased with antithymocyte globulin (ATG) therapy. T regulatory cells, as in other human immune-mediated diseases, are decreased in AA. IFN-γ and TNF negative effects on the proliferation of early and late hematopoietic progenitor, and stem cells are far more potent when these cytokines are secreted into the BM microenvironment than when they were simply added to in vitro cultures. IFN-γ and TNF can suppress hematopoiesis by inhibiting cell proliferation, inducing Fas-mediated apoptosis, and blocking hematopoietic growth factor intracellular signals. The early immune system events that must precede the global destruction of hematopoietic cells are not clear. Involvement of CD4 lymphocytes has been suggested based on the overrepresentation of HLA-DR15 among patients with immune-mediated AA. Clones of HLA-DR–restricted T cells derived from a few patients have been shown to proliferate in response to BM cells.
Many features of human AA can be reproduced in mouse models of GVHD, in which the donor inoculum lacks stem cells. Major and minor histocompatibility mismatches demonstrate the potency and specificity of small numbers of T cells, the role of cytokines, efficacy of immunosuppressive therapies, an “innocent bystander effect,” and roles for specific lymphocyte regulatory and effector T-cell subsets.
Radiation
BM aplasia is a major acute toxic effect of radiation ( Fig. 31.5 ); a dose-related pancytopenia occurs 2 to 4 weeks after exposure to radiation. Mortality from hematologic toxicity is a function of the ability of BM to tolerate damage to stem cells. The capacity for hematopoietic recovery after even massive single irradiation exposures is considerable, reflecting the resistance of the quiescent stem cell damage and enormous BM repopulating potential. At intermediate radiation doses around the median lethal dose (LD 50 ), at which BM toxicity limits survival, supportive efforts can dramatically alter outcomes. Autopsies of atomic bomb victims in Japan showed acellular BMs in the first weeks of the explosion, but later regenerating BM was frequently present. The histologic picture of radiation-mediated aplasia includes necrosis, nuclear pyknosis and karyorrhexis, nuclear lysis, and ultimately cytolysis; the associated phagocytosis, marked congestion, and hemorrhage are rapidly followed by fatty replacement. BM hypoplasia occurs with radiation doses higher than 1.5 to 2 Gy to the whole body. Precise LD 50 figures for humans do not exist, and estimates are based on limited direct human data and extrapolation from animal experiments. The LD 50 is highly dependent on the quality of medical care, and improved support may double the tolerated radiation dose. From assessment of the outcome of radiation accidents and high-dose therapeutic irradiation, the LD 50 has been estimated at approximately 4.5 Gy (see Fig. 31.5 ).
Although the immediate management of pancytopenia after a single large dose of irradiation is similar to that for treating AA, some unique points should be made concerning immediate evaluation and long-term prognosis. The type and intensity of the source of radiation, and the distance and shielding of the subject are the major determinants of radiation injury. However, these factors are often difficult to assess. Early recognition of the nature of the accident provides the best opportunity for dosimetry by accident reconstruction and use of blocking, displacement, or chelation agents. Exposure correlates well with the degree of pancytopenia. Because lymphocytes are particularly sensitive to radiation, their rate of decline can be used to estimate the dose of total body exposure to a level of approximately 3 Gy. At higher doses, the fall in the numbers of granulocytes and the severity of thrombocytopenia and reticulocytopenia can be used as gauges. The survival of some patients who received doses higher than 9 Gy suggests that autologous BM reconstitution may occur in most persons who survive the immediate consequences of accidental radiation exposure.
AA is not well documented as a delayed event after radiation exposure. A variety of hematologic abnormalities are associated with chronic low-level radiation exposure, most commonly lymphocytosis, neutropenia, dysmorphic leukocytes, and giant platelets (see Fig. 31.5 ). Cytogenetic abnormalities accumulate with time after chronic exposure, but they may not be reliably related to dose. AA does not appear to be more frequent among nuclear power plant or thorium processing factory workers. The excessive risk of death from AA previously reported after therapeutic irradiation of the spine for ankylosing spondylitis may have been overestimated. Cancer patients who had received therapeutic irradiation or higher exposure to natural background radiation were not found to have an increased risk of AA.
Drugs and Chemicals
AA is frequently associated with medical drug use ( Table 31.3 ). At the end of the 19th century, chemicals were linked to BM function through observations of benzene effects on workers. Establishment of a relationship between amidopyrine analgesics and agranulocytosis in the early 20th century and an apparent epidemic of AA after the introduction of chloramphenicol in the 1960s also supported this concept. Initially suggested by the accumulation of case reports, drug associations have been established in formal case-control population-based epidemiologic studies. In the IAAAS, relative risks were estimated for individual drugs and large classes of pharmaceutical agents, including nonsteroidal antiinflammatory drugs (NSAIDs), drugs affecting thyroid function, certain cardiovascular agents, some psychotropics, and sulfa-based antibiotics ( Table 31.4 ). Approximately 25% of the cases of AA identified in the IAAAS could be attributed to drug use. Drug use as a risk factor was also assessed by similar methods in Thailand, where the incidence of AA is higher than in the West. Surprisingly, chloramphenicol was not shown to be a risk factor; the etiologic fraction for drugs was only 15%.
Table 31.3
Classification of Drugs and Chemicals Associated With Aplastic Anemia
| I. Agents That Regularly Produce Marrow Depression as a Major Toxic Effect When Used in Commonly Used Doses or Normal Exposures |
|
| II. Agents Probably Associated With Aplastic Anemia but With a Relatively Low Probability Relative to Their Use |
|
| III. Agents More Rarely Associated With Aplastic Anemia |
|
Table 31.4
Drugs Associated With Aplastic Anemia in the International Aplastic Anemia and Agranulocytosis Study a
From Kaufman DW, Kelly JP, Levy M, Shapiro S. The Drug Etiology of Agranulocytosis and Aplastic Anemia . New York: Oxford University Press; 1991.
| Drug | Stratified Risk Estimate (95% CI) | Multivariate Relative Risk Estimate (95% CI) |
|---|---|---|
| Nonsteroidal Analgesics | ||
| Butazones | 3.7 (1.9–7.2) | 5.1 (2.1–12) |
| Indomethacin | 7.1 (3.4–15) | 8.2 (3.3–20) |
| Piroxicam | 9.8 (3.3–29) | 7.4 (2.1–26) |
| Diclofenac | 4.6 (2.0–11) | 4.2 (1.6–11) |
| Antibiotics | ||
| Sulfonamides b | 2.8 (1.1–7.3) | 2.2 (0.6–7.4) |
| Antithyroid drugs | 16 (4.8–54) | 11 (2.0–56) |
| Cardiovascular Drugs | ||
| Furosemide | 3.3 (1.6–7.0) | 3.1 (1.2–8.0) |
| Psychotropic Drugs | ||
| Phenothiazines | 3.0 (1.1–8.2) | 1.6 |
| Corticosteroids | 5.0 (2.8–8.9) | 3.5 (1.6–7.7) |
| Allopurinol | 7.3 (3.0–17) | 5.9 (1.8–19) |
| Gold | 29 (9.7–89) | |
CI , Confidence interval.
a The multivariate model included the following factors: age, gender, geographic area, date of interview, reliability of the patient, person interviewed, transfer from another hospital, history of blood disorder or tuberculosis, exposure to benzene and related chemicals, and use of other suspected drugs.
Associations between drug exposure and AA can be divided into two classes. Drugs used in cancer chemotherapy are selected for their cytotoxicity, and their regular, dose-dependent induction of BM aplasia is an expected effect. Most AA associated with medical drug use in the community is described as idiosyncratic, meaning that its occurrences are unexpected and rare. Many of the drugs implicated in AA also appear to cause other, milder forms of BM suppression such as neutropenia. Although difficult to prove, some dose relationship probably does exist even for idiosyncratic reactions. In most case reports, patients received normal or high doses of the agent, usually for a period of weeks to months. Drug-induced aplasia cannot be distinguished by history from idiopathic forms of the disease; the clinical course, including the favorable response to immunosuppressive therapy, is the same as in idiopathic disease.
The low probability of developing AA after a course of drugs may be a reflection of the gene variant frequency for metabolic enzymes (for direct chemical effects) or immune response genes. The rarity of idiosyncratic drug reactions could then arise from the infrequent combination of unusual circumstances: exposure, genetic variations in drug metabolism, the physical properties of the agent, enzymatic pathways that chemically alter the drug, and the susceptibility of the host to the action of a toxic compound. Examples of detoxifying enzyme systems directly applicable to BM failure that also demonstrate genetic variability include arylhydrocarbon hydroxylase (e.g., benzene toxicity), epoxide hydrolases (e.g., phenytoin toxicity), S -methylation (e.g., 6-mercaptopurine, 6-thioguanine, azathioprine) and N -acetylation (e.g., sulfa drugs). Genomic approaches have revealed the complex role of genetic variation in metabolic pathways that process arylhydrocarbons and even links to immune function.
Benzene
Benzene exposure is linked to AA. Benzene myelotoxicity can be placed between the predictable effects of chemotherapeutic agents and idiosyncratic drug reactions. Industrial emissions add greatly to the biologic sources of ambient benzene. Significant benzene exposure can also occur outside of industry. Although the concentrations of benzene to which consumers are exposed are orders of magnitude lower than those observed in industrial workers, the effect of low-dose chronic exposure is uncertain, but genetic variations in metabolizing enzymes may influence susceptibility to BM suppression at these levels. Benzene metabolites are also generated from the diet. Water-soluble products of benzene metabolism such as phenols, hydroquinones, and catechols mediate toxicity to the BM. Benzene and its intermediate metabolites covalently and irreversibly bind to BM DNA, inhibit DNA synthesis, and introduce DNA strand breaks. Benzene acts as a “mitotic poison” and as a mutagen. Acutely, the more mature, actively cycling BM precursor cells are preferentially damaged over more primitive progenitors. Intermittent exposure may be more damaging to the stem cell compartment than is continuous exposure. BM stroma can also be damaged by benzene.
The range of hematologic disease attributable to benzene is broad, from relatively frequent mild alterations in blood counts to AA or leukemia. Studies of exposed North American workers earlier in the 20th century suggested that the risk of AA was 3% to 4% in men exposed to concentrations higher than 300 ppm and that 50% of individuals exposed to 100 ppm developed some blood cell count depression. Leukopenia, anemia, thrombocytopenia, and lymphocytopenia are common consequences of benzene; other manifestations include macrocytosis, an acquired Pelger-Huet anomaly, eosinophilia, basophilia, and less often, polycythemia, leukocytosis, thrombocytosis, or splenomegaly. The BM is usually normocellular but can show hypocellularity or hypercellularity; a hypercellular phase can precede complete aplasia.
Aromatic Hydrocarbons
The common perception that other molecules resembling benzene or containing a benzene ring can also cause AA is not well supported by evidence. Neither the closely related alkylbenzenes nor pure toluene or xylene are established BM toxins. Often, an aromatic hydrocarbon has been implicated as causative by a clinician only for lack of another apparent etiology. For some substances, toxicity might result from the presence of benzene as a contaminant of the synthesis of the molecule or in the petroleum distillates used to dissolve the compound. However, the total number of AA cases reported with aromatic hydrocarbon exposures is small when the large populations exposed to this heterogeneous group of chemicals are considered. For example, the significance of a handful of case reports associated with insecticide exposure in the context of the vast use of these compounds is questionable. However, the very high prevalence of aromatic hydrocarbons in daily life would greatly amplify even a small individual risk. Pesticides and insecticides have been associated with AA for decades, with almost 300 medical case reports appearing in the medical literature. The most frequently cited insecticides are chlordane, lindane, and dichlorodiphenyltrichloroethane.
Chloramphenicol
The structural similarity of chloramphenicol to amidopyrine, a drug known to cause agranulocytosis, led to early prediction of possible hematotoxicity associated with the administration of this antibiotic. During the period of its unrestrained use, chloramphenicol was considered the most common cause of AA in the United States, accounting for 20% to 30% of total cases and 50% of drug-associated cases. Estimates of the risk of AA after a course of chloramphenicol ranged from 1 case per 20,000 to 1 case per 800,000 people. Based on these figures, a course of chloramphenicol was estimated to increase the risk of AA 13-fold. Although the introduction of chloramphenicol into the US market was perceived as having increased the total number of cases of AA, this assumption was only weakly supported by epidemiologic data, and the mortality from AA remained essentially constant during the period of chloramphenicol’s introduction, extensive use, and after the withdrawal of chloramphenicol from the market. Chloramphenicol has not been associated with AA in Thailand, despite its high rate of use there. In Hong Kong, where the use of chloramphenicol is almost 100 times higher than in the West, drug-associated AA occurs infrequently.
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