Acquired Aplastic Anemia and Pure Red Cell Aplasia PDF

C H A P T E R Acquired Aplastic Anemia and Pure Red Cell Aplasia CHAPTER OUTLINE APLASTIC ANEMIA Epidemiology Causal Factors Pathophysiology Clinical Evaluation Acquired and congenital bone marrow failure syndromes are characterized by a reduction in the effective produc- tion of mature erythrocytes, granulocytes, and platelets by the bone marrow. Bone marrow failure leads to various peripheral blood cytopenias. In some conditions, only one or two cell lines may be affected. In others such as aplastic anemia the result is pancytopenia. In this chapter, we deal only with the acquired bone marrow failure syndromes, though we use the term rather loosely because the acquired marrow failure syndromes may have a genetic basis. The known inherited/congenital bone marrow failure syn- dromes are described in detail in Chapter 7. The acquired marrow failure syndromes that affect only platelets or granulocytes are described in Chapters 22 and 34 respec- tively. The acquired single lineage deficiency syndromes that involve red cell production (pure red cell aplasia) are also described in this chapter, except that the 5Q- syndrome is discussed in Chapter 7 and in more detail Chapter 51. APLASTIC ANEMIA Decreased production of mature blood cells may result from a reduction in the number or function of their pro- genitors. Aplastic anemia is a descriptive term referring to a clinical state in which peripheral blood pancytopenia results from reduced or absent production of blood cells in the bone marrow. Aplastic anemia may arise in the setting of inherited/congenital syndromes associated with a predisposition to marrow failure (discussed in Chapter 7), may develop secondary to marrow-toxic stressors in an otherwise seemingly normal host, or may lack any apparent underlying cause. Classifications of the aplastic 162 SECTION II BONE MARROW FAILURE Box 6-1 Classification of the Aplastic Anemias339 ACQUIRED Secondary Radiation Drugs and chemicals Direct toxicity: chemotherapy; benzene Idiosyncratic: chloramphenicol; antiinflammatory leptics; carbonic anhydrase inhibitors Viruses Epstein-Barr virus Hepatitis (non-A, B, C, E, or G) Human immunodeficiency virus Immune diseases Eosinophilic fasciitis Hypoimmunoglobulinemia Systemic lupus erythematosis (uncommon) Thymoma Graft-versus-host disease in immunodeficiency Pregnancy Paroxysmal nocturnal hemoglobinuria Myelodysplasia Idiopathic INHERITED Fanconi anemia Dyskeratosis congenita Shwachman-Diamond syndrome Amegakaryocytic thrombocytopenia Diamond-Blackfan anemia Reticular dysgenesis GATA-2 syndromes Familial aplastic anemias Nonhematologic syndromes (e.g., Down, Dubowitz, syndromes) incidence of aplastic anemia have been obtained from smaller studies in the United States and earlier in Europe, but these figures may have been inflated by the inclusion of cases of myelodysplasia, a much more common syndrome. Aplastic anemia is more common in Asia (4 to 7 per million per year)4 than in the West. Chloramphenicol, a known cause of aplastic anemia, has been widely used in much of Asia because of its and low cost, but reductions in its use have not been accompanied by reductions in aplastic anemia incidence in Japan5,6 or elsewhere,7,8 and no association was observed in the case control studies in Thailand.9 case control study10 in Thailand spanning 1989-2002 described an association between aplastic anemia and exposure to benzene or pesticides. Although an risk of aplastic anemia was also associated with exposures and ingestion of nonbottled or nondistilled water, no significant associations with known or hepatitis were observed. The peak age of presentation of aplastic anemia 15 to 25 years or over 60 years. The male-to-female in acquired aplastic anemia is approximately 1 : 1. Causal Factors When no causative factors are ascertained, the cases are classified as idiopathic. A search for possible agents is warranted because some patients may improve following the removal of the offending agent. However, most cases of aplastic anemia remain idiopathic. The Box 6-2 Classification of Drugs and Chemicals Associated with Aplastic Anemia* AGENTS THAT REGULARLY PRODUCE MARROW DEPRESSION Antibiotics: daunorubicin, doxorubicin hydrochloride chloramphenicol Antimetabolites: antifolic compounds, nucleotide analogues Antimitotics: vinblastine, colchicine Benzene and chemicals containing benzene: carbon tetrachloride, chlorophenols, kerosene, Stoddard solvent Cytotoxic cancer chemotherapy with alkylating phalan, cyclophosphamide AGENTS POSSIBLY ASSOCIATED BUT WITH A LOW PROBABILITY RELATIVE TO USE Chloramphenicol Insecticides: chlordane, chlorophenothane (DDT), chloride (lindane), parathion Anticonvulsants: carbamazepine, hydantoins, Nonsteroidal antiinflammatory agents: indomethacin, phenylbutazone, phenylbutazone, sulindac Antihistamines: cimetidine, chlorpheniramine, ranitidine Antiprotozoal drugs: quinacrine, chloroquine Sulfonamides: some antibiotics, antidiabetics butamide), antithyroid drugs (methimazole, pylthiouracil), carbonic anhydrase inhibitors (acetazolamide, methazolamide) Penicillamine Metals: gold, arsenic, bismuth, mercury AGENTS MORE RARELY ASSOCIATED Allopurinol (may potentiate marrow suppression Antibiotics: flucytosine, mebendazole, methicillin, streptomycin, tetracycline, trimethoprim/sulfamet Carbimazole Guanidine Lithium Methyldopa Potassium perchlorate Quinidine Sedatives and tranquilizers: chlordiazepoxide, meprobamate, methyprylon, piperacetazine, prochlorperazine Thiocyanate Modified from Young NS, Alter BP: Aplastic anemia: acquired and inherited, Philadelphia, 1994, WB Saunders, p 104. *Agents are listed because they have been cited inclusion in this list does not imply acceptance causal relationship. become aplastic, five had sickle cell anemia and aplastic crises now known to be due to parvovirus infection.12 The incidence of drug-related aplasia in pediatric cases is low, mainly because many of the drugs felt to be related to aplasia are not used in childhood, with the exception of antiepileptic drugs, carbonic anhydrase inhibitors, nonsteroidal antiinflammatory medications, and some antibiotics. Drug-related aplasia may occur in several ways. Drugs may exert direct cytotoxic or suppressive effects on the bone marrow. Myelosuppressive drugs, such as those used in cancer chemotherapy, lead to predictable and dose-related marrow suppression. Benzene, too, can be demonstrated regularly to suppress the bone marrow in animals in a dose-linked manner, and most individuals exposed to sufficient amounts of benzene would suffer some type of marrow damage. In practice, most drug-related aplastic anemia is idiosyncratic and occurs unpredictably in only rare individuals who are prescribed 164 SECTION II BONE MARROW FAILURE derivatives, and petroleum products.59,60,61,62 tic anemia, leukemia, or both, have been reported years later in factory workers who had benzene exposure. Benzene is concentrated in bone marrow fat,63 forms water-soluble intermediates,64,65,66 and 67,68,69 DNA. It decreases the numbers of progenitors and damages stroma. The risk of cytopenias is likely related to cumulative exposure. Other Aromatic Hydrocarbons Toxicity thought to be due to other organic solvents may in some instances be caused by benzene contaminants. Neither pure toluene nor xylene is a marrow toxin. Aplastic anemia has been linked by many case reports to insecticides, particularly γ-hexachlorobenzene (lindane) in children.70 Aromatic hydrocarbons are present in insec- ticides and herbicides and may comprise the solvents for these agents. Some organophosphate insecticides have been shown to inhibit in vitro hematopoietic colony for- mation, as has lindane.71 Ionizing Radiation Marrow aplasia may occur as an acute toxic sequela of irradiation due to nuclear bomb explosion, radioactive fallout, reactor accidents, and accidental exposure in medicine and industry. Bone marrow cells may be affected by high-energy γ-rays as well as by ingested or absorbed lower-energy α particles. The radiation injury is to the actively replicating pool of precursor and progenitor cells and also to stem cells, in which DNA damage may have a more severe effect. Nonetheless, radiation-related marrow aplasia is infrequent. Even in a restricted episode, such as the Chernobyl reactor accident in 1986, most immediate deaths were due to skin burns and damage to gastrointestinal and pulmonary systems. Chronic radiation-induced aplasia is dose related. Patients who were irradiated for ankylosing spondylitis had an increased risk of aplastic anemia, and American radiologists have been reported to have an increased death rate from aplasia (for both groups, the distinction of aplasia and myelodysplasia was not made). The incidence of late aplasia in atomic bomb victims was not increased, nor was it increased in patients receiving radiation therapy for malignancies. Knospe and co-workers suggested that irradiation with an expo- sure greater than 4.4 Gy was required for the develop- ment of aplasia; they also postulated that low doses might damage only stem cells, whereas high doses would also damage the supportive hematopoietic stromal microenvironment.72 Infectious Agents Patients with bacterial or viral illnesses frequently develop mild pancytopenia during or following the infec- tion (see Chapter 37). Patients with bacterial or viral infections often receive antibiotics and other medica- tions, and it is frequently not clear whether an ensuing aplastic anemia was caused by the infection, by the drug, or by the combination of the two, or even whether the infectious illness was the result and not the cause of the pancytopenia. factors;88 direct progenitor cell infection by some CMV strains also has been documented.88,89 Herpesvirus 6 is the cause of exanthem subitum90 and, like CMV, may be found in the marrow of patients with graft failure after transplantation,91 as well as in hematopoietic progenitors infected in vitro.92 As with other viruses, both of these are ubiquitous infections making causality difficult to ascertain. Human Immunodeficiency Virus Patients with acquired immunodeficiency syndrome (AIDS) often have cytopenias,93 but their marrow is much more commonly cellular and dysplastic than empty. Colony formation by marrow from patients may be diminished.94,95,96,97 The action of human immunodefi- ciency virus-1 (HIV-1), a lentivirus, on hematopoietic cells remains a subject of controversy. HIV-1 infection of CD34+ cells has been difficult to detect in vivo from patient material96,98 or after tissue culture infection of normal cells.96,99 The virus apparently directly infects megakaryocytes, which bear the CD4 receptor present on T cells.100 The virus also may affect stroma functions, at least in vitro.101,102 HIV-1 can act indirectly on hemato- poiesis through inhibitory lymphokine production: The envelope glycoprotein can stimulate macrophages to produce tumor necrosis factor (TNF), which in turn inhibits hematopoietic colony formation.103 Hematologic suppression can also be due to opportunistic infections, neoplasms, or marrow suppression from the drugs used to treat AIDS and its complications. Other Viruses Blood count abnormalities, which are rarely severe, may be observed in the course of rubella, measles, mumps, varicella, and influenza A.104 Paroxysmal Nocturnal Hemoglobinuria Paroxysmal nocturnal hemoglobinuria (PNH) is a disease characterized by variable combinations of mild to severe intravascular hemolysis, large venous thromboses, and aplastic anemia.105 It is uncommon in adults and even rarer in children (see Chapter 13). There is a clear asso- ciation of PNH with aplastic anemia: Many patients with PNH develop pancytopenia and marrow hypoplasia, and PNH clones are detectable in up to 50% to 70% of patients with acquired aplastic anemia.106,107,108,109 PNH is characterized by an inability to inactivate complement on the erythrocyte cell surface, resulting in increased sensitivity to complement. Deficits were subse- quently identified in a family of membrane proteins, all of which were anchored to the cell membrane via glyco- sylphosphatidylinositol (GPI). GPI binds covalently to specific carboxyl terminal protein sequences and attaches them to cell membrane phosphatidylinositol residues. The genetic defect in PNH was localized to the X-linked phosphatidylinositol glycan class A (PIG-A) gene, whose product functions in the transfer of N-acetylglucosamine to phosphatidylinositol as an early step in GPI anchor formation. Early tests for PNH, such as the Ham test or sucrose hemolysis test, relied on the demonstration of increased sensitivity to complement-mediated red cell 166 SECTION II BONE MARROW FAILURE of the GPI-negative cells in PNH or aplastic anemia is currently unclear.124 Despite the presence of clonal popu- lations of PNH cells, patients with PNH do not exhibit an increased incidence of leukemias, and the clones do not behave in a malignant fashion. Immunologic Diseases Ten percent of patients with the rare collagen syndrome, eosinophilic fasciitis, have associated anemia.125 This condition is usually limited Thymoma associated with hematopoietic failure gener- ally but not exclusively presents as pure red Some of the adult cases had also received drugs such as chloramphenicol or sulfonamides, and the aplasia could have been due to the drugs alone or to the combination An iatrogenic aplasia can be induced by the transfusio of competent lymphocytes into immunodeficient hosts. In these cases, marrow failure arises from the transfusio graft-versus-host disease (GVHD).127 Pathophysiology The purported mechanisms for failure of hematopoiesis are predicated on our current knowledge of normal hematopoiesis (see Chapter 7). Disease could decreased numbers or defective function of the soluble components required for blood cell production from exogenous factors that result in damage tion of hematopoietic cells. Figure 6-2 depicts potential mechanisms for the loss of hematopoietic in aplastic anemia. In model I, marrow toxins such as irradiation, drugs, or chemicals or direct invasion by viruses might cause hematopoietic stem and/or progeni- tor cell death. In model II, an underlying abnormality the hematopoietic cells may result in a predisposition hematopoietic stem cell damage, premature stem or insufficient stem cell production (see Chapter model III, viruses, drugs, toxins, or immune tion may incite a cellular or humoral immunologic tion against a normal hematopoietic compartment. In model IV, abnormalities of the marrow stromal environ- ment may actively inhibit hematopoiesis. These possibili- ties are not mutually exclusive, and supportive data for each model have been described. Current data suggest that aplastic anemia may represent the final seemingly common outcome of different pathogenic mechanisms, with different underlying causes entailing different medical management considerations. Hematopoietic Stem Cells and Clonality Patients with aplastic anemia have decreased numbers of blood and marrow committed progenitor cells, assayed as myeloid colony-forming cells (CFU-GMs); erythroid colony-forming cells (CFU-Es and burst- forming units erthyroid [BFU-Es]); megakaryocytic pro- genitors (CFU-megas); multipotent colony-forming cells (CFU granulocyte, erythrocyte, monocyte, megakaryo- cyte [CFU-GEMMs]); and long-term culture-initiating cells (LTC-ICs).128,129 Hematopoietic cells can also be assessed phenotypically for the presence of the CD34 antigen, which defines a compartment of about 1% of marrow cells that includes progenitors and possibly permanent disorders of the bone marrow environment, including immunologic mechanisms, as the cause of stem cell destruction. Low stem cell numbers persist even with hematologic recovery after medical therapy with antithy- mocyte globulin (ATG) and cyclosporine (see following discussion), and blood cell parameters, such as macrocy- tosis, do not return to normal in many cases, again suggestive of an underlying stem cell abnormality. A primary stem cell defect likely also contributes to the observation of clonal hematopoiesis in aplastic anemia140 as well as late development of other hematologic diseases such as myelodysplasia and acute leukemia (see discussion). A subset of patients with seemingly idiopathic aplastic anemia may actually carry an inherited/congenital predis- position to marrow failure (see Chapter 7). Although the inherited/congenital marrow failure syndromes are often associated with characteristic physical findings, patients may present with isolated aplastic anemia as the sole manifestation of their underlying inherited syndrome.141 In some patients lacking significant physical findings or cytopenias at the time of evaluation, an elevated mean corpuscular volume (MCV) may be the only clue to an underlying inherited marrow failure syndrome. A careful family history for hematologic abnormalities, malignancy predisposition, or other characteristic clinical stigmata may be helpful in distinguishing an inherited marrow failure syndrome.142 For example a subset of patients presenting with aplastic anemia without other clinical stigmata of an inherited marrow failure syndrome were found to harbor mutations in the dyskeratosis congenita genes hTERC,143,144 which encodes the RNA component of telomerase, or hTERT,145,146,147 which encodes the cata- lytic subunit of the telomerase complex. Telomeres are specialized structures that stabilize the ends of each chro- mosome to prevent excessive shortening with replication, to distinguish chromosome ends from internal DNA breaks, and to prevent end-to-end fusions. Telomeres consist of six–base pair repeated sequences (TTAGGG) associated with a specific protein complex. Telomerase is a ribonucleoprotein enzyme consisting of an H/ACA RNA component (TERC) complexed with a reverse tran- scriptase (TERT) and additional H/ACA binding proteins including dyskerin, GAR1, NHP2, and NOP10. When telomerase levels are diminished or absent, telomeres pro- gressively shorten with each cell division. At a critically short telomere length, a checkpoint is triggered for cells to stop dividing and undergo senescence to prevent chro- mosomal rearrangements. Shortened telomeres have been observed in the leuko- cytes of aplastic anemia patients.148 Of five patients noted to have mean telomere lengths less than 5 kb, three had acquired cytogenetic abnormalities, suggesting that com- promise in the telomere’s probable role in stabilizing chromosome ends may contribute to the increased inci- dence of myelodysplasias and acute leukemias. A study by Brummendorf and colleagues reported relatively normal telomere lengths in patients who responded to immunosuppression, while untreated or unresponsive patients showed significant telomere shortening.149 Telo- mere shortening is a common feature associated with 168 SECTION II BONE MARROW FAILURE has been reported166 and may suggest that such agents might exert additional effects on the bone marrow.167 Nonetheless multiple reports have demonstrated an association of aplastic anemia with alterations in lym- phocyte numbers or specific immunologic changes at presentation and in some cases further changes with hematologic responses. Hoffman and colleagues showed inhibition of erythroid growth in vitro utilizing coculture of peripheral blood lymphocytes from five out of seven aplastic anemia patients studied. 168 Zoumbos and col- leagues noted increased circulating and bone marrow levels of IFN-γ in aplastic anemia patients and a response of increased colony growth in vitro in response to inhibi- tion with neutralizing antibody to this proinflammatory cytokine.169 Hinterberger and colleagues failed to confirm the reported increased circulating levels of proinflamma- tory cytokines in aplastic anemia patients, but onstrate increased TNF and IFN-γ production by peripheral blood mononuclear cells (PBMCs) from aplas- tic anemia patients at baseline and after stimulation in vitro.170 Risitano and colleagues reported a dominant clonal T-cell immune response characterized by flow cytometry using antibodies against different TCR-β sub- families and by sequencing the CDR3 region of the TCR-β chain in 54 patients studied with aplastic anemia.171 The size of these clones and the total number of T cells decreased following successful treatment with ATG and increased with relapse. This phenomenon was associated with HLA-DR1501 genotype and activated cytotoxic T cells assayed in vitro. The association with HLA type was not confirmed by a subsequent study of Japanese patients.172 Solomou and colleagues reported reduced T regulatory cells (TRegs) (CD4+, CD25+, FoxP3+) in 20 patients with aplastic anemia that was consistent with increased IFN-γ and a TH1 polarization phenotype of T cells.173 Six of seven patents showed slightly increased numbers of TRegs after immunosup- pression therapy (IST) during hematologic responses. In a case report, Ikeda and colleagues noted marked altera- tion of the CD4/CD8 T-cell ratio after hepatitis that was subsequently followed by onset of aplastic anemia.174 IST was followed by normalization of lymphocyte subsets with hematologic recovery. These studies imply concomitant immune dysregula- tion with aplastic anemia and suggest a mechanism by which IST could be working therapeutically. However, no specific antigen has previously been implicated mech- anistically in the evolution of aplasia. In a recent report, it was shown that a prodrug of the anti-HIV drug, aba- cavir, which is associated with a hypersensitivity reaction in treated patients, can bind to the HLA-B*57:01 mole- cule, resulting in altered peptide presentation.175 The altered peptides appear as foreign and lead to activation of CD8+ T cells. One can imagine such a scenario leading to acquired aplastic anemia if the abnormal peptide pre- sentation of “self-antigens” leads to CD8 activation directed against hematopoietic stem cells in the bone marrow. While such a finding is not reported yet in aplas- tic anemia patients, this could underlie the immunologic mechanism long hypothesized as the etiology of this disease. Interestingly Weinstock and colleagues also months ex vivo.190 Subsequently Marsh and colleagues examined LTMC from 32 adult aplastic anemia patients and found 31 of the 32 had abnormal hematopoiesis in these cultures.191 However, using cross-over cultures, only 1 of 23 patients had defective adherent components. Juneja and colleagues demonstrated reduced cobblestone areas (containing active hematopoiesis) but normal adher- ent cell fractions in eight aplastic anemia patients com- pared with normal controls.192 Marsh and colleagues utilized human LTMC and assessed the HM in cross-over experiments.193 They demonstrated that stroma generated from aplastic anemia patients performed normally when inoculated with CD34+ cells from normal donors. Nissen and colleagues compared short-term stroma and progeni- tor colony growth in aplastic anemia patients treated with or without IST and found a modest increase in both of these cell fractions in treated versus untreated patients.194 Chatterejee and colleagues used a cytotoxic drug animal model of aplasia to demonstrate failure of recovery of marrow stromal cells with time.195 While this study may be relevant to some isolated instances of acquired aplastic anemia, it seems more relevant to marrow damage in cancer or leukemia patients undergo- ing therapy. Shipounova and colleagues showed impaired adipogenesis and osteogenic potential with increased size and number of fibroblast colonies from aplastic anemia patients’ bone marrow.196 These authors also demon- strated increased stromal-derived cytokines, including ANG1, VEGF, and VCAM-1. Overall these studies suggest some alterations in the HM of aplastic anemia patients, but there is currently no definitive evidence in humans of a mechanistic link between defective marrow microenvironment and development of acquired aplastic anemia. More recently interest has focused on defining specific anatomic and/or biochemical spaces (niches) in the bone marrow occupied by hematopoietic stem cells. Schofield first termed the phrase stem cell niche to define a sup- porting cellular environment in which hematopoietic stem cells reside.197 Currently controversy exists about the overall relevance of the two best-studied stem cell niches.198 Hematopoietic stem cells appear to reside in close proximity to osteoblasts that line the cortical bone of the marrow space, and osteoblasts may be important in maintaining quiescence of the primitive hematopoietic stem cells. This area of the marrow has thus been termed the osteoblastic niche. A subset of these cells, spindle- shaped N-cadherin+ CD45− osteoblasts (SNO cells) resid- ing in trabecular bone, may be derived from mesenchymal stem cells, and their number correlate with the content of long-term hematopoietic stem cells.199 Ablation of osteoblasts leads to loss of HSC200 and failure of hema- topoiesis, while manipulations that increase osteoblasts have the opposite effect.201 An anatomically distinct putative hematopoietic stem cell niche is made up in part by sinusoidal endothelia cells and has been termed the perivascular niche.202 Some data suggest this niche may be critical for mobilization and homing of hematopoietic stem cells via the Rac GTPase pathway203,204 and/or for progenitor cell prolif- eration and differentiation,205,206 although these studies 170 SECTION II BONE MARROW FAILURE Box 6-3 Causes of Pancytopenia HYPOCELLULAR BONE MARROW Acquired aplastic anemia Inherited bone marrow failure syndrome (Fanconi anemia, amegakaryocytic thrombocytopenia, dyskeratosis congenita, Shwachman–Diamond syndrome, Paroxysmal nocturnal hemoglobinuria* Hypoplastic myelodysplastic syndrome Virus-associated aplastic anemia CELLULAR BONE MARROW PRIMARY BONE MARROW DISEASE Myelodysplasia Paroxysmal nocturnal hemoglobinuria* SECONDARY TO SYSTEMIC DISEASE Systemic lupus erythematosis, Sjögren syndrome Hypersplenism Vitamin B12, folate deficiency Infection Storage disease (Gaucher, Niemann-Pick) Alcoholism Sarcoidosis Infiltrative bone marrow disorders Acute myelogenous leukemia Acute lymphoblastic leukemia Hemophagocytic lymphohistiocytosis Metastatic solid tumors Osteopetrosis Myelofibrosis activity, or exercise intolerance, though the gradual onset of severe anemia can be surprisingly well compensated in young children. Serious infection is not a frequent pre- senting symptom early in the course of aplastic anemia. Many patients feel well with few symptoms on initial presentation. A history of steatorrhea or fatty food intol- erance may be a manifestation of Shwachman–Diamond syndrome, though the absence of these symptoms does not rule out this diagnosis.210 Gastrointestinal disorders may also be associated with other inherited marrow fail- ures syndromes such as dyskeratosis congenita or Fanconi anemia. A careful family history for blood disorders, malignancies (particularly ovarian and breast cancer or hepatocellular carcinoma), premature graying, hepatitis, pulmonary fibrosis, or congenital anomalies that might suggest an inherited bone marrow syndrome should be sought. Patients with a history suggestive of a familial marrow failure syndrome or familial MDS should be evaluated further with genetic and laboratory studies. A developmental history can provide a helpful screen for potential congenital, metabolic, or storage diseases. A detailed medication, environmental, and infectious history, particularly for the period 1 to 12 months prior to presentation, should be obtained. Physical Examination Clinical appearance is related to the severity and duration of the underlying pancytopenia. Hemorrhagic manifesta- tions from thrombocytopenia occur early and include petechiae (particularly on the border of the hard and soft palate), ecchymoses, epistaxis, and bleeding from mucous TABLE 6-1 Clinical Evaluation340 Clinical History Recent illnesses Medications Toxic exposures Family history of blood disorders, malignancy, Physical Examination Vital signs Height and weight Congenital anomalies, stigmata Laboratory Tests Complete blood count and differential Morphology Reticulocyte count Bone marrow biopsy Bone marrow aspirate Morphology Cytogenetics Culture Other Peripheral blood Chemistry AST, ALT, GGT, Bilirubin, LDH Coombs test BUN, creatinine, electrolytes Serologic testing/PCR Flow cytometry for CD55/CD59 Diepoxybutane or mitomycin C chromosomal breakage Telomere length, dyskeratosis congenita Autoimmune disease evaluation Histocompatibility testing of patient and family ALT, Alanine aminotransferase; AST, aspartate dehydrogenase. both qualitatively and quantitatively. The bone marrow in aplastic anemia is hypocellular, with empty spicules, and increased fat, reticulum cells, plasma cells, and mast cells. In young children there may be residual lymphocytes that appear prominent due to the lack of myeloid elements in the marrow cavity. Some dyseryth- ropoiesis, with megaloblastosis and nuclear-cytoplasmic 172 SECTION II BONE MARROW FAILURE aspirates should also be sent for karyotype analysis and fluorescence in situ hybridization (FISH) to assess for clonal cytogenetic abnormalities associated with myelo- dysplasia or malignancy. While FISH studies are more sensitive, they can only detect the specific chromosomal abnormality tested. Bone marrow may also be sent for culture or DNA-based antigen detection of infectious agents such as viruses, though interpretation is tempered by the frequent persistence of viral DNA long after the active infection has passed. Management and Outcome Prognosis Outcome depends on the types of treatments offered, the causes of the aplasias, and the eras and countries analyzed. In a large series conducted before 1957, Wolff found a survival rate of only 3%.214 In other series ending no later than 1965, complete recoveries were reported in 10% to 35% of patients. In a series of 40 pediatric patients seen before 1958, Shahidi and Diamond found that only two patients recovered, one of whom later had recurrence,215 though these studies may have included patients with moderate disease for whom spontaneous recovery is more common.216,217 Long-term prognosis is adversely affected by delayed complications such as clonal disease. The clinical course of 24 pediatric patients with moderate aplastic anemia was followed for a median of 66 months (range 10 to 293 months).218 Moderate aplas- tic anemia was defined as marrow hypoplasia of less than 50%, at least two cytopenias such as an absolute neutro- phil count (ANC) less than 1500/mm3, platelet count less than 100,000/mm3, or reticulocytopenic anemia lasting at least 6 weeks. Sixteen patients (67%) progressed to severe aplastic anemia, five patients (21%) remained in the moderate range, and three patients (12%) resolved. It is currently unclear whether early institution of therapy for moderate aplastic anemia confers improved outcome. Because current therapies are associated with significant side effects, their institution for children with moderate aplastic anemia, for whom the marrow aplasia may spon- taneously resolve, warrants careful consideration. Spontaneous Recovery Spontaneous recovery has been reported anecdotally, although most cases were probably of moderate rather than of severe aplastic anemia. Good supportive care (see following discussion) may contribute to this; 14 of 33 children recovered,219 and children with moder- ate aplastic anemia have been found to have an excellent prognosis even without specific treatment.220 A study of immunosuppressive therapy in which 21 patients were randomized (some to receive only supportive care) found no improvement in the control group after 3 months.221 Spontaneous recovery is sufficiently rare in severe aplastic anemia that all modalities of current therapy should be initiated. Supportive Care Blood product support should be used judiciously to avoid sensitization, and blood product donors should infected locus is often difficult. The bacterial organisms may be gram-negative bacilli such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, as well as gram-positive cocci, including Staphylococcus aureus and S. epidermidis and streptococci. Use of G-CSF has been studied in a few prospective analysis and other randomized trials, and these studies have been examined by meta-analysis. Gluckman and colleagues randomized aplastic patients receiving IST to prophylactic G-CSF treatment or not.231 These investigators reported enhanced recovery of neutrophils in the G-CSF group but no effect on overall survival. More recently, Tichelli and colleagues randomized 192 aplastic patients to ATG/cyclosporine +/− G-CSF and reported significantly fewer infectious episodes and hospitalization days in the G-CSF group but no differences in overall survival or event-free sur- vival between groups.230 Interestingly they found lack of neutrophil response at day 30 post initiation of treatment was significantly associated with a lower response rate overall and a lower survival rate. They postulated that if confirmed by additional studies, the lack of neutrophil recovery at 30 days posttreatment could identify a group of patients in which alternative treatment might begin earlier than in the past. did not confirm a prior study by Teramura and col- leagues that reported a lower incidence of relapse in patients treated with G-CSF.232 Meta-analysis of a large number of studies has also reported that in aggregrate these studies showed no effect of G-CSF on overall sur- vival, hematologic responses, or specific infections and no association of the use of G-CSF with MDS, AML or PNH.233 Immunosuppression due to preparation for BMT or as primary therapy for the aplastic anemia may lead to unusual bacterial, fungal, viral, and protozoan infections. Recommendations for specific antibiotics and other anti- infection agents are beyond the scope of this chapter, and regimens are changing rapidly as new generations of treatments are developed. The use of routine prophylactic antibiotics has no demonstrated role in the “well” patient with aplastic anemia. In many institutions, guidelines are derived from experience in neutropenic cancer and post–BMT patients because prospective studies in pediatric aplastic anemia patients are rare.229 In the opinion of many, even without supporting outcomes data, aplastic anemia patients with ANC less than 500 should receive antibacterial and anti- fungal prophylaxis. Patients undergoing IST are typically given prophylaxis for Pneumocystis carinii, although data supporting the need for this treatment are sparse or nonexistent. In the context of fever and neutropenia, complete evaluation and cultures of all possible sites should be followed by the administration of broad- spectrum parenteral antibiotics. Fungal infections occur frequently in neutropenic patients who have received repeated or extended courses of antibiotics; candidiasis and especially aspergillosis, which lead to sinusitis, lung disease, or disseminated infection, are distressingly fre- quent causes of death in aplastic anemia.228 Antecedent or concurrent administration of steroids increases this risk. Aggressive introduction of antifungal treatment is 174 SECTION II BONE MARROW FAILURE granulocyte counts in addition to the lymphocyte counts, and it may lead to a positive Coombs test. Thrombocy- topenia may require intensive blood product support during this time.238 Responses to ATG given as a single agent usually occur within the first 3-months, if at all, and blood count improvement by 3 months correlates with survival.239 The earliest response to therapy may be heralded by the appearance of a few granulocytes and nucleated red cells in the circulation. Red-cell size distribution histograms typically show a macrocytic shoulder.240 Reticulocytes appear, transfusions decline, and the hemoglobin level increases slowly. The white cell count rises next. The last cell line to return is often the platelets, whose numbers may remain low for months or even years. The recovering red cells are not normal; in addition to macrocytosis, Hb F level is increased, and fetal membrane antigens may remain present.241 Long-term survivors (except those with successful transplants) often have residual abnormalities, such as thrombocytopenia, red cell macrocytosis, and ele- vated Hb F levels.242 Clearly we do not understand either the mechanism of action of this poorly characterized drug, the ultimate course of patients with ATG-induced remis- sions, or the basis of their partial recovery. Cyclosporine. Cyclosporine (formerly cyclosporin A) is a fungal cyclic undecapeptide and an effective immu- nosuppressive agent.243 Among its many effects, cyclospo- rine can inhibit T cells, preventing production of IL-2 and IFN-γ.160 About one half of patients who had failed ATG or ALG treatment subsequently experienced remission with cyclosporine.244,245 Treatment with cyclosporine alone yielded significantly lower response rates and higher rates of disease progression as compared with combined therapy.246,247 Oral cyclosporine should be administered twice daily to maintain blood trough levels between 100 and 250 ng/ mL as measured by radioimmunoassay, though the target levels may vary depending on laboratory methods used to determine cyclosporine levels. Hematologic responses can take weeks to months, and an initial trial period of 3 to 6 months is generally recommended. Toxic effects from cyclosporine use are not insignifi- cant and include hypertension, hypertensive encepha lopathy, azotemia, hirsutism, gingival hypertrophy, coarsening of facial features, tremor, immunodeficiency, increase in serum creatinine levels, transient or irrevers- ible nephrotoxicity, hepatotoxicity, seizures, and P. carinii pneumonia.243 As with ATG, in vitro tests of the effect of cyclosporine on progenitor cells do not predict responses.248 Combination Immunosuppressive Therapy. The combi- nation of ATG with cyclosporine has resulted in higher rates of hematologic recovery than either drug alone.246,247,249 Importantly, intensive IST has greatly improved the results of therapy in two groups commonly refractory to ALG or ATG alone: children and patients with very severe disease.250,251 In the National Institutes of Health (NIH) Clinical Center trial,252 survival curves of those older and younger than 35 years of age were similar. A large study including both children and adults TABLE 6-2 Pediatric Studies of Immunosuppressive Patient Study Years Number Treatment Fuher 1993-1997 86 (49vSAA, ALG, et al255 30SAA, CSA, 7MAA) G-CSF* Kojima 1992-1997 119 (50 ATG, et al250 vSAA, 36 CSA, SAA, 33 DAN, MAA) +/-G- CSF Kojima 1992-1997 113 ATG, et al278 (39vSAA, CSA, 47SAA, DAN, 27MAA) +/- G-CSF Goldenberg 1990-2003 14 ATG, CSA et al257 +/-G- CSF Fuher 1993-2001 97 vSAA ATG CSA et al251 G-CSF 49 SAA CR, Complete response; CSA, cyclosporine A; DAN, partial response; SAA, severe aplastic anemia; *G-CSF added for SAA and vSAA. most studies, failure of response is typically defined as persistence of severe aplastic anemia. In adult patients failing the first treatment with horse ATG, 63% of patients receiving a second course of horse ATG responded,263 while 77% (23 out of 30) of patients initially treated with horse ATG and cyclosporine responded to a second course with rabbit ATG and cyclosporine.264 In some small studies in children, the response rate to second immunosuppressoin has been less,265 and treatment with rabbit ATG as a second therapy was associated with only 27% response rate.266 In a large NIH study that included 77 children less than 18 years of age, of 14 relapsed patients, 6 were retreated with ATG and were alive at report, but the hematologic response of these patients was not reported.261 Less acute reactions were noted when the source of ATG was varied between the first and second treatments. The time frame to response following a second course of immunosuppression was similar to that observed following initial treatment. Patients failing to respond to two courses of immunosuppressive therapy are unlikely to respond to a third treatment course.267 Because androgens are effective in treating some patients with aplastic anemia (see following discussion), the effect of combining androgen treatment with ATG was examined in several trials. Champlin and colleagues 176 SECTION II BONE MARROW FAILURE between studies. An EBMT study including both children 6 Akiko Shimamura and David A. Williams Management and Outcome PURE RED CELL APLASIA Primary Acquired Pure Red Cell Aplasia Secondary Acquired Pure Red Cell Aplasia anemias are presented in Box 6-1. A careful medical history, physical examination, and laboratory evaluation are critical to discern inherited versus acquired causes of aplastic anemia. The distinction between inherited and acquired causes of aplastic anemia carries profound implications for medical management and treatment, so a careful search for possible underlying inherited syn- dromes should be undertaken prior to initiation of therapy. Camitta and co-workers classified the severity of aplas- tic anemia in an effort to make possible the comparison of diverse groups of patients and different therapeutic approaches.1 Diagnosis of severe aplastic anemia requires that the patient have at least two of the following anoma- lies: a granulocyte count below 500/µL, a platelet count below 20,000/µL, and an absolute reticulocyte count less than or equal to 40 × 109/L. In addition the bone marrow biopsy must contain less than 25% of the normal cellular- ity or less than 30% hematopoietic elements (Fig. 6-1). Very severe aplastic anemia is further defined by a granu- locyte count of less than 200/µL.2 Mild or moderate aplastic anemia, sometimes called hypoplastic anemia, is distinguished from the severe form by the presence of mild or moderate cytopenias and more variable, but still deficient, bone marrow cellularity. These distinctions are more than semantic; they are critical for the prediction of outcome and the choice of therapy. Epidemiology Epidemiologic studies performed in Europe estimate that the annual incidence of aplastic anemia is 2 per million per year.3 By comparison, the incidence of acute leukemia is about 50 per million per year. Higher figures for the 161 distinction between acquired versus inherited causes of aplastic anemia is crucial to guide clinical management and treatment (see Chapter 8). Drugs The incidence of drug- and chemical-related aplastic drugs; antiepi- anemia11 varies over time and from place to place. Many drugs and toxins have been implicated by inferential and circumstantial evidence; the magnitude of the risk is usually unknown (Box 6-2). Presence of an agent on this list suggests caution regarding its use, but no drug on this list should be proscribed if there are strong clinical indica- tions for its use. From a public health perspective, even drugs associated with an increased risk of marrow failure do not cause large numbers of cases of aplastic anemia.3 Note that even confirmed associations do not substan- tiate causality. Antibiotics felt to be causative may have been administered for the viral infection that led to the aplastic anemia or for symptoms from already established neutropenia in an undiagnosed case of aplastic anemia. Bleeding may be precipitated in undiagnosed thrombocy- topenic patients who receive nonsteroidal antiinflamma- tory drugs. As an example of known errors in associations, among six patients reported to have sniffed glue and and Seckel surveys efficacy A A recent increased animal infections is at ratio B causative Figure 6-1 Bone marrow examination in severe aplastic anemia. A, Note hypocellularity in biopsy. B, Residual cells seen in aspirate are lymphocytes, stromal cells, plasma cells, and mast cells. (Courtesy of Dr. Gail Wolfe, Boston, Mass.) 6 Acquired Aplastic Anemia and Pure Red Cell Aplasia 163 the medication, sometimes weeks to months after its administration is discontinued. This last category of patients may possess a genetic propensity for this phe- nomenon or be due to a direct effect of the drug on (Adriamycin), antigen presentation of abnormal peptides (see below). Chloramphenicol Chloramphenicol was considered to be the commonest cause of aplastic anemia at the peak of its use, which drugs: busulfan, mel- began in 1949.13 A genetic predisposition may exist. The mechanism of the idiosyncratic aplasia remains unknown despite extensive investigation. Chloramphenicol contains a nitrobenzene ring and thus resembles amidopyrine, a drug known to cause agranulo- γ-benzene hexa- cytosis.14,15 Chloramphenicol is the prime example of a phenacemide drug that causes both dose-related marrow suppression ibuprofen, oxy- through mechanisms that include mitochondrial inhibi- tion,16 and idiosyncratic aplastic anemia.17 The signs of dose-related toxicity appear more rapidly in patients with hepatic or renal disease because the drug (chlorpropamide, tol- methylthiouracil, pro- must be inactivated by conjugation with glucuronide in the liver and excreted in the urine. High doses and high plasma levels correlate with the characteristic reversible erythroid depression. In vitro, chloramphenicol inhibits the growth of both colony-forming units granulocyte/ macrophage (CFU-GMs) and colony-forming units ery- by cytotoxic drugs) throid (CFU-Es)18,19,20,21,22 and also may inhibit the hema- sulfonamides, topoietic microenvironment (HM).20,23 hoxazole Other Drugs Nonsteroidal antiinflammatory drugs, which are used more extensively in adults than in children, are associated with aplasia.3 Nonsteroidal antiinflammatory drugs asso- chlorpromazine, ciated by occasional case reports with aplastic anemia include aspirin,24 indomethacin,25,26,27,28 and ibuprofen.29 Several large studies reported an increased risk of aplastic anemia with phenylbutazone30,31 and identified even higher probabilities with some of the other nonsteroidal in the literature; antiinflammatory drugs.3 Cimetidine, another commonly by the author of a used drug, is associated with a 2 per 100,000 user risk of cytopenias.32,33 Sulfa-containing compounds, which appear as risk factors in most case-control studies of drugs and aplastic anemia, are used in a wide variety of clinical circumstances.34,35,36,37,38,39,40,41,42,43 Other drugs implicated in aplastic anemia that are commonly used in the pediatric population include anticonvulsants (hydantoins,44,45,46 carbamazepine47,48,49,50,51) and carbonic anhydrase inhibi- tors52 (acetazolamide53,54 and methazolamide55,56). Many of the drugs listed in Figure 6-1 also have been associated with agranulocytosis. In general only a minority of cases of aplastic anemia can be assigned a drug association. The distinction between aplasia secondary to the medica- tion versus aplasia arising from the underlying disorder (or occult viral infection) requiring treatment can be difficult. Chemicals and Toxins probably Benzene Benzene is a particularly dangerous environmental con- taminant.57,58 It is found in organic solvents, coal tar Fatal aplas- Hepatitis Although hepatitis is frequently associated with mild depression of blood cell counts, aplasia is a rare sequela, damages estimated to occur in fewer than 0.07% of the total number of pediatric hepatitis cases73 and in fewer than 2% of those with non-A, non-B hepatitis.74 Nonetheless as an identifiable clinical event, a prior episode of hepa- titis is recognized in 2% to 5% of aplastic anemia patients in a Western series.75 The prevalence of prior hepatitis is about twofold this proportion in the Far East.76 Among children with aplastic anemia in Taiwan, 24% had a history of recent acute hepatitis.77 Antecedent hepatitis may be subclinical, as about 50% of patients with aplas- tic anemia may have elevated hepatic transaminases before their first transfusion. In a report of 32 patients with liver transplantation for hepatic failure following non-A, non-B hepatitis, 28% developed aplastic anemia.78 While aplasia has been reported following both hepatitis A and B virus infections,79, 80 the majority of cases of the hepatitis/aplasia syndrome are not associated with sero- typable hepatitis virus.81, 82 A study of aplastic anemia patients reported to the European Registry from 1990-2007 found that 5% of patients with aplastic anemia had an antecedent serone- gative hepatitis. Hepatitis-associated aplastic anemia was slightly more common in males and tended to present at a younger age. Actuarial survival after treatment with either immunosuppression or hematopoietic stem cell transplant was similar between patients with hepatitis- associated aplastic anemia versus other acquired aplastic anemia.83 Flaviviruses Flaviviruses cause arbovirus hemorrhagic fevers, dengue, and other hematodepressive syndromes. Dengue can propagate in bone marrow cultures without direct cyto- toxicity, and dengue antigens induce lymphocyte activa- tion and the release of marrow suppressive cytokines.84 pathologic Epstein-Barr Virus Herpesviruses such as Epstein-Barr virus (EBV) are large, complex DNA viruses.85 EBV causes infectious mono- nucleosis, which has pancytopenic complications in less than 1% of cases. More than 12 such cases have been reported, and one half of these cases had a fatal outcome. In one study, EBV was demonstrated by immunologic and molecular methods in the bone marrow of six patients with aplastic anemia.86 Only two had a history of typical mononucleosis, although all six had serologic evidence, suggesting that EBV may be an unrecognized cause of aplastic anemia. The EBV’s target is B cells, although T cells also may be infected. Because EBV is a common infection, issues of ascertainment can render the causative determination of aplasia difficult. Cytomegalovirus and Human Herpesvirus 6 Infection with cytomegalovirus (CMV) may lead to graft failure in immunosuppressed bone marrow transplant (BMT) recipients.87 CMV can infect marrow stromal cells in vitro and can inhibit their ability to produce growth 6 Acquired Aplastic Anemia and Pure Red Cell Aplasia 165 lysis. Increased sensitivity and specificity are achieved using flow cytometry assays for the absence of GPI- anchored proteins such as CD59 and CD55.110 More sensitive tests utilize aerolysin, a channel-forming toxin that binds GPI-anchored proteins to result in cell lysis but leaves the GPI-deficient PNH cells intact.111 The use of a fluorescently labeled aerolysin variant that binds to GPI but fails to lyse the cells offers increased sensitivity and specificity for the detection of the PNH clone.112 The clinical significance of small populations of PNH clones is unclear, and most of the published data are derived from adult patients. Small numbers of PNH cells are detectable in healthy controls,113 although the PNH cells in this context are typically polyclonal. Despite the frequent finding of PNH clones in the bone marrows of aplastic anemia patients, only 10% to 15% of patients subsequently develop the clinical syndrome of PNH.114 In the majority of patients, such clones may persist or disappear; however, some patients may develop large symptomatic PNH clones requiring therapeutic interven- tion.115,116 Patients with small asymptomatic PNH clones may respond to the same treatments as other patients with aplastic anemia. Patients who develop large PNH clones are at risk for developing symptoms of hemolysis or thrombosis.115 One recent retrospective study included 47 pediatric patients with aplastic anemia of whom 14 had a PNH clone greater than 1% at baseline (median clone size 16%). The median clone size remained small for all patients, and none of these patients developed clinical symptoms of PNH.115 The etiology of marrow aplasia in PNH remains an area of active investigation. Although severe combined immunodeficiency (SCID) mice infused with bone marrow from PNH patients show preferential engraftment with the PNH clones,117 studies of hematopoiesis in PNH patients have not detected any selective proliferative advantage of the PIG-A− hematopoietic clones.118 A subsequent study comparing in vitro proliferation of PIG-A− and PIG-A+ CD34 cells from PNH patients found a selective growth deficiency in the PIG-A+ cell population rather than an advantage for the PIG-A mutant cells: Fas expression was elevated on the wild type compared with the GPI-deficient cells, suggesting increased resistance to apoptosis as one potential mecha- nism to explain their findings.119 No proliferative advan- tage of PNH hematopoietic clones was observed in mice mosaic for the PIG-A gene.120,121 To evaluate the hypoth- esis that autoreactive T cells might preferentially elimi- nate PIG-A+ hematopoietic stem cells while sparing the PNH clones, the sensitivity of normal versus PNH EBV- transformed B-cell lines to autologous EBV-specific T-cell lines was examined.122 The PNH cells were no less sensi- tive to T-cell-mediated cytotoxicity than the non-PNH cells; thus the GPI-linked cell surface molecules are not required for killing by T cells. An abnormal distribution of expanded T-cell clones detected by size analysis of the complementarity-determining region 3 (CDR3) in the β-variable region (BV) mRNA of the T-cell receptor (TCR) has been noted in PNH patients,123 although the targets of such T-cell populations remain to be ascer- tained. The mechanisms underlying the clonal expansion I Toxins medications infections GPI-negative Normal HSC vascular aplastic to adults. II cell aplasia.126 ? Marrow stress? . n Abnormal HSC nal III T T T T result from cellular or or or elimina- Immune attack different cells IV of to cell loss, 1). In dysregula- reac- Abnormal marrow microenvironment Figure 6-2 Models for the pathogenesis of aplastic anemia. I. Healthy hematopoietic stem cells and progenitors are damaged by exogenous agents such as toxins, medications, or infectious pathogens resulting in marrow aplasia. II. Abnormal marrow cells (e.g., inherited marrow failure syndromes) undergo premature attrition. Marrow aplasia might be exacerbated by external factors. III. Immune-mediated attack (cel- lular or humeral) eliminates hematopoietic stem cells and progenitors. IV. Abnormal marrow microenvironment impairs hematopoiesis. HSC, Hematopoietic stem cell. activated stem cells.130,131,132,133 CD34+ cells are reduced in aplastic anemia, and almost all patients show not only severe reduction in the numbers of these cells but also poor plating efficiency for colony formation from purified CD34+ cells.134,135 Several lines of evidence point to primary abnormali- ties in the hematopoietic stem cell as a potential etiology for aplastic anemia. Multiple studies have noted that simple infusion of stem cells from an identical twin donor without prior conditioning was sufficient to treat the aplasia in many cases.136,137,138,139 These results strongly support a pathogenic stem cell defect and argue against 6 Acquired Aplastic Anemia and Pure Red Cell Aplasia 167 marrow failure, though telomere lengths are particularly short in patients with dyskeratosis congenita, a disorder of telomerase function, even in comparison to other marrow failure patients.150 Whether telomere shortening in marrow failure patients lacking telomerase mutations represents a primary defect in telomere maintenance or a secondary effect of increased cycling of the few remaining hematopoietic stem cells remains to be clarified. Shorter telomere lengths have been associated with a higher risk of relapse and clonal progression with decreased survival in patients with aplastic anemia.151 following Immune Destruction Several lines of data suggest that immune phenomena may result in aplastic anemia.152 In some syngeneic (iden- tical twin) BMTs, simple infusion of stem cells is not sufficient to reconstitute hematopoiesis. The requirement for prior conditioning of the host may reflect some opera- tional immunologic process, though it is also consistent with the presence of an underlying clonal abnormality that requires ablation.153 Histocompatibility studies con- ducted in Europe, Asia, and America have indicated an increased representation of HLA-DR2 among aplastic anemia patients, consistent with a genetically determined immune susceptibility to disease.153,154,155 Evidence for specific immune mechanisms is reviewed below. Lymphocytes Lymphocyte infusions in mouse models result in marrow destruction and aplastic anemia.156 Autologous recovery has been reported after either mismatched or matched transplants in patients prepared with antilymphocyte sera or cyclophosphamide.157,158,159 While the etiology of aplastic anemia in children remains unclear, evidence sup- ports an immunologic mechanism of disease in the absence of known environmental toxins, drugs, and con- stitutional syndromes. Overriding these considerations are the large proportion of responses in acquired aplastic anemia to immunosuppressive therapies, although the antilymphocyte preparations contain heterogeneous mix- tures of antibodies to many cells, including lymphocytes, and are clearly immunosuppressive as well as generally cytotoxic. Cyclosporine exerts inhibitory T cells effects and inhibits transcription of genes for cytokines including IL-2 and interferon-gamma (IFN-γ).160 It has many other toxic effects on several tissues, including brain and kidney. The diverse antibodies in ATG react with a wide range of antigens, including signal transduction and adhesion molecules.161 Thus the attribution of immune dysregula- tion as causal in aplastic anemia based on therapy must be tempered with caution because ATG and cyclosporine exert multiple effects in addition to immunosuppression. Of note, therapies with mixtures of monoclonal antibod- ies specific for human T cells have not been effective in clinical trials to date162 (see following discussion). Fur- thermore ATG can increase colony growth in normal, myelodysplastic syndrome (MDS), and aplastic anemia CD34+ bone marrow cells in culture.163,164 ATG treat- ment also reduces the expression of Fas-Ag on aplastic anemia bone marrow CD34+ cells.165 Effective treatment of myelodysplasia with immunosuppressive medications recently demonstrated somatic alterations in 20% of adult patients with acquired aplastic anemia utilizing next generation sequencing technology.176 Alterations included mutations of DNMT3A and ASCL1, both asso- ciated with MDS in adults, and mutations of β2- microglobulin, which is required for class I expression and CD8 T-cell function. Thus previously unrecognized mutations may predispose to acquired aplastic anemia via either mechanisms leading to a prodrome of myelo- dysplasia or by subtle alternations in immune functions. Limitations of these studies include the paucity of puri- fied cell populations required for sequencing and the lack of functional data that addresses the biologic relevance of these alterations with respect to stem cell function and hematopoiesis. Additional studies will undoubtedly follow. did dem- Lymphokines Whether overproduction or dysregulated production of inhibitory cytokines represents a primary etiology versus a secondary effect of an underlying bone marrow abnor- mality remains unclear. IFN-γ, a soluble inhibitor pro- duced by T cells, was found to be produced at high levels in cultures from aplastic anemia patients.177,178,169 IFN-γ is expressed in the marrow of most patients with aplastic anemia but not in normal people or in patients with other hematologic diseases who have undergone frequent trans- fusions.179,180 High pretreatment intracellular γ-interferon levels correlated with response to immunosuppressive therapy.181 Other inhibitory cytokines, such as TNF182 and macrophage inflammatory protein-1,183,184 also are overexpressed in aplastic marrow. Both interferon and TNF directly and synergistically inhibit hematopoiesis in vitro.185 In long-term bone marrow culture, constitutive low-level expression of IFN-γ is sufficient to markedly reduce the output of committed progenitor cells and LTC- ICs, the stem cell surrogate.186 Both interferon and TNF increase the potential for programmed cell death within the CD34+ compartment by increasing Fas antigen expression on target cells.187 Fas antigen is a cell surface molecule in the TNF receptor family; its activation signals apoptosis. Microenvironment In theory aplastic anemia could be due to a microenviron- ment that fails to support hematopoiesis: a lesion of “soil” rather than “seed.”72 The role of the HM in etiol- ogy of aplastic anemia or the response to treatment remains controversial. Ershler and colleagues showed decreased erythroid growth in whole bone explants from a single patient with constitutional red cell aplasia but no effect from the patient’s plasma, mononuclear cells, or T cells, implying an HM defect.188 This observation is remi- niscent of the HM defect seen in Steel mice,189 which lack production of normal stem cell factor (SCF) in nonhema- topoietic cells, but it seems more likely that this was simply a demonstration of a cell-intrinsic erythroid cell defect. Dexter and colleagues described an in vitro long- term marrow culture (LTMC) that consisted of an adher- ent cell compartment made up of a number of different cell types that supported hematopoiesis for weeks to 6 Acquired Aplastic Anemia and Pure Red Cell Aplasia 169 have not examined long-term hematopoietic stem cells in functional assays. Alterations of the sinusoidal endothe- lium with antibodies to VE-cadherin leads to failure of megakaryocytopoiesis in murine models.205 Some models suggest that quiescent hematopoietic stem cells may migrate from the osteoblastic niche to the perivascular niche via β1 integrin function and chemokine/growth factor stimulation. Overall, whether there are truly dis- tinct functional differences between these putative hema- topoietic stem cell niches remains to be determined, and the role of specific growth factors, adhesion molecules, and chemokines are incompletely understood. A long- term goal of investigators in this field is to recapitulate the HM in vitro such that true expansion of hematopoi- etic stem cells may occur in the laboratory setting. Success in this area would have a major impact in BMT and regenerative medicine. Hematopoietic growth factor production and plasma levels are usually increased rather than decreased in patients with aplastic anemia.207 Circulating levels of erythropoietin, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin, and flt-3 ligand are elevated in patients with aplastic anemia. SCF levels have been reported to be low to normal. Levels of interleukin 1 (IL-1), produced by monocytes, may be low in aplastic anemia. Therapeutic trials with these factors have yielded divergent and incomplete responses,208 casting doubt on the pathophysiologic significance of deficiency of these factors. The report of inhibitory effects of marrow adi- pocytes on hematopoiesis in mouse models is intriguing given the typical fatty replacement observed in aplastic bone marrows.209 Clinical Evaluation The differential diagnosis of pancytopenia is broad (Box 6-3). The evaluation and diagnostic work up of the patient with presumed acquired aplastic anemia should be directed at eliminating alternative diagnoses for which curative therapies are available or for which the therapies for aplastic anemia would be inappropriate (Table 6-1). Because the inherited marrow failure syndromes entail different medical management issues, a careful medical history, family history, physical examination, and appro- priate laboratory evaluation to assess for underlying inherited bone marrow failure syndromes should be pursued. The diagnosis of an inherited marrow failure syndrome also carries profound implications for hemato- poietic stem cell donor selection from family members and may be important for family planning. Given the wide clinical variability of the inherited marrow failure syndromes even amongst members of a given family, all siblings of a patient with an inherited marrow failure disorder should be tested whether or not they appear to carry clinical stigmata of the disease. l History The chief presenting symptoms relate to low blood counts. Bleeding, such as gum oozing, nosebleeds, easy bruising with minimal trauma, or heavy menses may be seen. Chronic anemia may present with fatigue, decreased membranes. Neutropenia may be associated with oral ulcerations, bacterial infections, and fever; these signs are rarely present early. Evidence of erythropoietic failure characterized by pallor, fatigue, and tachycardia is often late because the life span of the erythrocyte (120 days) far exceeds that of platelets (10 days) or white cells (vari- GATA-2 syndromes) able, but measured in hours for granulocytes). Mucous membranes and nail beds may be pale. Lymphadenopa- thy, splenomegaly, and severe weight loss are uncommon and may suggest other underlying disorders. Short stature, congenital anomalies (particularly of the thumbs and forearms), areas of hyper- or hypopigmentation, dystro- phic nails, immunologic abnormalities, or pulmonary disease should alert the examiner to possible inherited bone marrow failure disorders. Immunologic abnormali- ties or lymphedema raises consideration of inherited GATA-2 syndromes (see Chapter 24). Laboratory Studies Evaluation should include a complete blood count and reticulocyte count. A careful evaluation of the periph- eral blood smear may suggest infection or dietary defi- ciency. Blood counts are most often uniformly depressed. The blood smear shows a paucity of platelets and leuko- cytes. The anemia is often macrocytic and particularly useful in pointing to a possible constitutional syndrome, though if erythropoiesis is entirely arrested, it may be normocytic. The red cell distribution width (RDW), a numeric measure of anisocytosis, is most often normal. Platelet size is not increased, as it is in most cases of immune thrombocytopenias. The absolute reticulocyte count is decreased. Granulocyte numbers are clearly diminished, as may be those of monocytes and lympho- cytes.211,212 Increases in fetal hemoglobin (Hb F) and red cell i antigen are manifestations of the fetal-like erythro- poiesis seen in stress hematopoiesis213 but occur irregu- larly in aplastic anemia patients, have no prognostic value, and can persist in recovered patients. Laboratory evaluation for possible infectious etiolo- gies should be obtained as clinically indicated. Additional testing for possible immunologic or autoimmune diseases may be considered. Blood tests to rule out Fanconi anemia and PNH are important, as these diagnoses would entail alternative treatments. Telomere length measurements may be useful as a screen for possible underlying telomere disorders. Very short telomeres in long-lived lymphocyte populations should prompt consideration of additional genetic workup for dyskeratosis congenita. Data on the use of telomere measurements in isolation to guide clini- cal treatment decisions in this setting are sparse. A growing array of genetic testing is available to assist in the diagnosis of many of the inherited marrow failure syndromes (see Chapter 7). Patients with stigmata of col- lagen vascular disease should be appropriately evaluated. Early HLA typing of the patient and family members is helpful in guiding future therapies. Baseline renal and hepatic laboratory evaluations are also indicated prior to the initiation of potentially nephrotoxic or hepatotoxic therapies. Bone marrow examination must be done by both aspi- ration and biopsy so that cellularity can be evaluated 6 Acquired Aplastic Anemia and Pure Red Cell Aplasia 171 Rationale Infectious etiology Marrow suppression Marrow suppression congenital anomalies Assess for inherited marrow failure Clinical sequelae of cytopenias Assess for inherited marrow failure Assess for inherited marrow failure Assess for other systemic diseases Defines severity Malignant versus benign Vitamin B12 deficiency Storage disease Defines severity Differentiate production versus destruction Assess cellularity Assess architecture (granuloma, fibrosis, hemophagocytosis, infiltrative or metastatic disease) Malignant versus benign Storage disease Hemophagocytosis Congenital disorder Myelodysplasia Infectious agent (e.g., tuberculosis, virus) DNA/antigen-based viral tests Hepatitis Autoimmune cytopenias Chronic renal failure Hepatitis, EBV, HIV, other virus Paroxysmal nocturnal hemoglobinura Fanconi anemia Evidence of collagen vascular disease Establish potential donor pool aminotransferase; BUN, blood urea nitrogen; GGT, γ-glutamyl transferase; LDH, lactate asynchrony, may be seen. Aspirates alone may appear hypocellular owing to dilution with peripheral blood, or they may look hypercellular because of areas of focal residual hematopoiesis. Biopsies provide more reliable specimens for assessment of cellularity. Biopsies also provide critical information regarding bone marrow architecture such as fibrosis or granulomas. Bone marrow never be from the patient’s family for the same reason. Complete red cell phenotyping should be considered where feasible to minimize the potential risk of erythro- cyte antibodies in patients who may receive long-term blood product support. All blood products should be irradiated because standard treatments are associated with profound immunosuppression. Leukoreduction appears prudent to reduce the potential risk of infection transmission and allosensitization because aplastic anemia patients may require a hematopoietic stem cell transplant. Exposure to potentially hazardous drugs or toxins should be eliminated. Treatment with single hematopoietic growth factors, such as granulocyte-colony stimulating factor (G-CSF) or erythropoietin should be avoided in part due to the resulting delay in definitive therapy.222 being Bleeding Platelet (and red cell) support has probably had the great- est impact on survival in aplastic anemia and has changed the cause of death from bleeding to infection. Platelets can be provided from several individual units of blood or, preferably, by platelet pheresis from a single donor to reduce antigenic exposure (see Chapter 36). The main role of prophylactic platelet support is reduction of the risk of intracranial hemorrhage, which is a rare but potentially devastating event. The general threshold platelet count has traditionally been 20,000 cells/µL; however, this figure and the entire practice of prophylactic administration of platelet transfusions have recently been questioned.223,224,225 Platelet transfusion at platelet counts below 10,000/µL in stable outpatients with chronic severe aplastic anemia was feasible and safe.226 Chronic platelet transfusions for aplastic anemia are generally given when there are symptoms of bleeding or if the patient is at increased bleeding risk (e.g., toddlers learning to walk, patients with hypertension while on cyclosporine, patients with fever and infection). In some institutions a higher platelet transfusion trigger point of 20,000/µL is used during initiation of IST. Other measures to reduce bleeding include mainte- nance of good dental hygiene, use of a soft toothbrush, stool softeners, and avoidance of trauma. Drugs that may interfere with platelet function, such as aspirin or nonste- roidal antiinflammatory drugs, should be avoided in thrombocytopenia. Antifibrinolytic agents such as amino- caproic acid and tranexamic acid may decrease bleeding, particularly from the gums and nasal mucosa. in one series, Infections. There are few reported studies of infection in aplastic anemia.227,228 Severe granulocytopenia may last for years, and infection remains the leading cause of death in aplastic anemia patients, particularly bacterial and fungal infections.229,230 However, the immune systems of granulocytopenic patients remain intact; this is in con- trast to the situation of patients with malignancies who receive chemotherapy and who experience simultaneous neutropenia and immunosuppression (as during IST for aplastic anemia). Neutropenia (and perhaps monocyto- penia) increases the risk of bacterial infection in aplastic anemia.211 Because neutropenia precludes the develop- ment of an inflammatory response, identification of an 6 Acquired Aplastic Anemia and Pure Red Cell Aplasia 173 indicated in the persistently febrile patient who is unre- sponsive to antibiotics or in the appropriate clinical setting. Anemia. Red cells should be provided as clinically indi- cated. The hemoglobin should be maintained at a level consistent with normal activities if routine transfusions are to be used. Chronic anemia can be well tolerated once adaptation has occurred, and some children who can sustain a hemoglobin level greater than 6 to 7 g/dL without transfusions (i.e., a child who is not bleeding) may be carefully observed. Long-term transfusions lead to iron overload, with accumulation in critical organs. Permanent damage to heart, liver, and endocrine glands from iron overload may be prevented by iron chelation therapy.234 that the Treatment Hematopoietic Stem Cell Transplantation. Hematopoi- etic stem-cell transplant offers curative therapy for aplas- tic anemia. The general topic of BMT is described in Chapter 8. The role of BMT in aplastic anemia treatment is discussed in Chapter 8. This study Immunosuppression Antithymocyte Globulin. Antilymphocyte globulin (ALG) and ATG (the sera produced by immunization of horses or rabbits with human thoracic duct lymphocytes or thymocytes) are highly cytotoxic reagents with activity against all blood and marrow cells, including progenitors. These reagents were initially used to decrease the rate of rejection of HLA-matched bone marrow. ALG and/or cyclophosphamide were also employed to permit at least temporary engraftment with mismatched marrow in aplastic patients without a matched donor.157,158,159,235,236 Autologous recoveries occurred in some of these patients; they encouraged several groups to examine the use of ATG or ALG with and without haploidentical marrow. Treatment with ALG was superior to controls in both response rate and 3-year survival.221 The addition of addi- tional agents to ALG therapy can further enhance thera- peutic effect. ATG or ALG/ALS is often given to a total cumulative dose of 100 to 160 mg/kg, typically divided over 4 days. Immediate allergic reactions to ALG are rare and can be predicted by skin testing followed by desensitization in those who are allergic.237 The patient begins to make antibodies to horse protein about 1 week after first expo- sure; at this point, equine antibodies are rapidly cleared from the circulation. This reduces the effective dose and enhances immune complex formation. Serum sickness due to immune complex deposition may manifest around 11 days after initiation of treatment as fever, a serpigi- nous rash at the volar–dorsal border of the hands and feet, arthralgia, myalgia, lassitude, and changes in urine sediment. Bronchospasm or liver chemistry abnormalities may also occur. A short course of corticosteroids is usually given (prednisone, 1 mg/kg) beginning at the start of ATG treatment and for 10 to 14 days to reduce ATG- related side effects. ATG binds to determinants on all cell types in blood and marrow and reduces platelet and at the NIH reported a response rate of 67% at 43 months and a 5-year actuarial survival rate of 70% (86% for responders and 16% for nonresponders).253 The Gruppo Italiano Trapianti di Midolio Osseo (GITMO)/European Group for Blood and Marrow Transplantation (EBMT) study of 100 patients with severe aplastic anemia includ- ing children (median age 16 years) treated with ALG, cyclosporine, prednisolone, and G-CSF found trilineage hematopoietic response in 77% after one or more courses of ALG.254 Several trials in children have reported the efficacy of combined ATG and cyclosporine in the pedi- atric population with an overall response rate of close to 80%255,256,250,251,257 (Table 6-2). Although greater lympho- cyte depletion is achieved with rabbit ATG as compared to that with horse ATG, a randomized trial comparing horse ATG versus rabbit ATG in the upfront treatment of aplastic anemia demonstrated that superior hemato- logic response (68% versus 37%) and survival (96% versus 76%) was achieved with horse ATG.258 ATG is typically given over a 4-day course in combina- tion with a 6 to 12 month course of cyclosporine. The lack of a unified definition for response complicates com- parison of different studies, but generally a complete response (CR) refers to normal or functionally normal blood counts, a partial response (PR) denotes hemoglobin and platelet counts adequate to avoid the need for trans- fusion, and neutrophil counts typically greater than 500/µL, and no response commonly refers to continued severe aplastic anemia. Improvements in blood counts are delayed and may not be evident for many weeks. In a study including both children and adults, response rates, defined as at least a PR in two blood cell lineages, were 67% at 3 months and 71% at 6 months following initia- tion of combined ATG and cyclosporine therapy.252 Cyclosporine should be tapered gradually with close monitoring of the blood counts prior to each decrease in dosage. A persistent drop in the blood counts can often be treated with either an increase in cyclosporine dosage or reinitiating the full dose. Some patients require con- tinuous cyclosporine, and these patients should be main- tained on the lowest effective dose. Relapse risk was reduced when cyclosporine was tapered slowly.259 In a review of ongoing trials by the German multicenter group and the NIH, 29% and 14% of initial responders, respec- tively, were found to require continuous treatment.253 The reoccurrence of frank pancytopenia generally requires a second course of ATG followed by full-dose cyclosporine. The relapse rate after combined IST varies widely from study to study. Most studies do not include only children. Kamio and colleagues recently reported a relapse rate of 16% in 264 responding aplastic patients260 (which com- pared to 33% reported in an NIH study).261 Relapse rates were even lower in a Japanese study.232 Patients treated with IST often continue to have subnormal counts and may remain macrocytic. While the majority of patients respond within 6 months, delayed responses beyond that time may be seen for some patients. Around 20% of patients in pediatric studies fail to respond to ATG and cyclosporine therapy (Table 6-2), while a recent study of mostly adult patients reported failure of response in around 40% of patients.262 In 6 Acquired Aplastic Anemia and Pure Red Cell Aplasia 175 Therapy for Aplastic Anemia Follow-Up Clonal (years) Response Survival Relapse Abnormality 4 80% (OR) 87% 15% 3% clonal abnormalities 4.7% AML 4 71% (OR) 83% vSAA 13% vSAA 3% vSAA 91-93% 29% 61%(OR) MAA+SAA MAA/ SAA+G- SAA CSF, 83% +G-CSF (OR) 64% SAA-G- MAA/ CSF SAA -G-CSF Median 5.3 30% CR 87% NR 13.7% Range 31% PR cumulative 3.75-8.9 42% NR incidence at 6 MDS at 8 months years, 21.9% cumulative incidence clonal cytogenetic abnormalities at 8 years Median 4.4 93% (OR) 93% 0 0 Range 0.8-13.3 Median 4.1 69% (CR) 93% vSAA 13% vSAA NR vSAA 44% (CR) 81% SAA 14% SAA NR SAA danazol; MAA, moderate aplastic anemia; NR, not reported; OR, overall response; PR, vSAA, very severe aplastic anemia. reported no difference in response rate or survival in patients with moderate to severe aplastic anemia.268 Studies by Kaltwasser and colleagues269 and the EBMT270 found significantly higher response rates after ALG plus androgens (73% and 56%) than in patients receiving ALG alone (31% and 40%). A recent retrospective study in adults reported an overall response rate of 77% and overall survival rate of 78% at 5 years following treat- ment with ATG plus androgens.271 At least one report showed increased relapse after ATG and cyclosporine when danazol was included in the treatment.260 Because some patients with inherited causes of aplastic anemia may experience hematologic improvement following androgen therapy,272 it is important to rule out an under- lying inherited marrow failure syndrome. Androgen side effects may be unacceptable to some patients, and their use in acquired aplastic anemia in children is generally limited. Relapse. Relapse after immunosuppressive therapy has been reported in around one third of patients in studies including both children and adults,262,273 although lower rates of relapse may be attained with slow tapering of cyclosporine and continued maintenance doses at the lowest required cyclosporine dose for some patients. Lack of standardized definitions of relapse hamper comparison clonal transformation of stem cells may underlie aplas

Acquired Aplastic Anemia and Pure Red Cell Aplasia PDF

Document Details

Tags

Related

Summary

Full Transcript

Upgrade to continue