Anemia Aplastik

463

CHAPTER 34

APLASTIC ANEMIA: ACQUIRED AND INHERITED

George B. Segel and Marshall A. Lichtman

SUMMARY

Acquired aplastic anemia is a clinical syndrome in which there is a deficiencyof red cells, neutrophils, monocytes, and platelets in the blood, and fattyreplacement of the marrow with a near absence of hematopoietic precursorcells. Reticulocytopenia, neutropenia, monocytopenia, and thrombocytopenia,when severe, are life-threatening because of the risk of infection and bleeding,complicated by severe anemia. Most cases occur without an evident precipitatingcause and are the result of the expression of autoreactive cytotoxic T lympho-cytes that suppress or destroy primitive CD34+ multipotential hematopoieticcells. The disorder also can occur after (1) prolonged high-dose exposure tocertain toxic chemicals (e.g., benzene), (2) after specific viral infections (e.g.,Epstein-Barr virus), (3) as an idiosyncratic response to certain pharmaceuticals(e.g., ticlopidine, chloramphenicol), (4) as a feature of a connective tissue orautoimmune disorder (e.g., lupus erythematosus), or, (5) rarely, in associationwith pregnancy. The final common pathway may be through cytotoxic T-cellautoreactivity, whether idiopathic or associated with an inciting agent sincethey all respond in a similar fashion to immunosuppressive therapy. The differ-ential diagnosis of acquired aplastic anemia includes the hypoplastic marrowthat can accompany paroxysmal nocturnal hemoglobinuria or hypoplastic oli-goblastic (myelodysplastic syndrome) or polyblastic myelogenous leukemia.Allogeneic hematopoietic stem cell transplantation is curative in approximately80 percent of younger patients with high-resolution human leukocyte antigen(HLA)-matched sibling donors although the posttransplant period may beseverely complicated by graft-versus-host disease. The disease may be signifi-cantly ameliorated or occasionally cured by immunotherapy, especially a regi-men coupling antithymocyte globulin with cyclosporine. However, aftersuccessful treatment with immunosuppressive agents, the disease may relapseor evolve into a clonal myeloid disorder, such as paroxysmal nocturnal hemo-globinuria, a clonal cytopenia, or oligoblastic or polyblastic myelogenous leu-kemia. Several uncommon inherited disorders, including Fanconi anemia,Shwachman-Diamond syndrome, dyskeratosis congenita and others have as aprimary manifestation aplastic hematopoiesis.

ACQUIRED APLASTIC ANEMIA

■

DEFINITION AND HISTORY

Aplastic anemia is a clinical syndrome that results from a marked diminu-tion of marrow blood cell production. The latter results in reticulocytope-nia, anemia, granulocytopenia, monocytopenia, and thrombocytopenia.The diagnosis usually requires the presence of pancytopenia with a neutro-phil count fewer than 1500/

μ

L (1.5

×

10

9

/L), a platelet count fewer than50,000/

μ

L (50

×

10

9

/L), a hemoglobin concentration less than 10 g/dL (100g/L), and an absolute reticulocyte count fewer than 40,000/

μ

L (40

×

10

9

/L),accompanied by a hypocellular marrow without abnormal or malignantcells or fibrosis.

1

For the purpose of therapeutic decision making, compara-tive clinical trials, and international sharing of data, the disease has beenstratified into moderately severe, severe, and very severe acquired aplasticanemia based on the blood counts (especially the neutrophil count) andthe degree of marrow hypocellularity (Table 34–1). Most cases of aplasticanemia are acquired; fewer cases are the result of an inherited disorder,such as Fanconi anemia, Shwachman-Diamond syndrome, and others (see“Hereditary Aplastic Anemia” below).

Aplastic anemia was first recognized by Ehrlich in 1888.

2

Hedescribed a young, pregnant woman who died of severe anemia andneutropenia. Autopsy examination revealed a fatty marrow with essen-tially no hematopoiesis. The name

aplastic anemia

was subsequentlyapplied to this disease by Chauffard, a French hematologist, in 1904,

3

and although an anachronistic term because the morbidity is the resultof pancytopenia, especially neutropenia and thrombocytopenia, thedesignation is entrenched in medical usage. For the next 40 years, manyconditions that caused pancytopenia were confused with aplastic ane-mia based on incomplete or inadequate histologic study of the patient’smarrow.

4

The development of improved instruments for percutaneousmarrow biopsy in the last half of the 20th century improved diagnosticprecision. In 1972, Thomas and his colleagues established that marrowtransplantation from a histocompatible sibling could cure the disease.

5

The disease initially was thought to result from an atrophy or chemicalinjury of primitive marrow hematopoietic cells. The unexpected recov-ery of marrow recipients who were given immunosuppressive condi-tioning therapy but who did not engraft with donor stem cells raisedthe possibility that the disease may not be intrinsic to primitive hema-topoietic cells but the result of a suppression of hematopoietic cells byimmune cells, notably T lymphocytes.

6

The requirement to treat therecipient of a marrow transplant from an identical twin with immuno-suppressive conditioning therapy for optimal results of transplant, but-tressed this suspicion.

7

This supposition was confirmed by a clinicaltrial that established antilymphocyte globulin (ALG) capable of amelio-rating the disease in the majority of patients.

8

Since that time, compel-ling evidence for a cellular autoimmune mechanism has accumulated(see the main section “Etiology and Pathogenesis” below).

■

EPIDEMIOLOGY

The International Aplastic Anemia and Agranulocytosis Study and aFrench study found the incidence rate of acquired aplastic anemia to beabout 2 per 1,000,000 persons per year.

1,9

This approximate annualincidence rate has been confirmed in studies in Spain (Barcelona),

10

Brazil (State of Parana),

11

and Canada (British Columbia).

12

The high-est frequency of aplastic anemia occurs in persons between the ages of15 and 25 years; a second peak occurs between the ages of 65 and 69.

1

Aplastic anemia is more prevalent in the Far East where the incidence isapproximately 7 per 1,000,000 in parts of China,

13

approximately 4 per1,000,000 in sections of Thailand,

14

approximately 5 per 1,000,000 inareas of Malaysia,

15

and approximately 7 per 1,000,000 among childrenof Asian descent living in a province of Canada.

12

The explanation for a

Acronyms and abbreviations that appear in this chapter include: A, adenine;ALG, antilymphocyte globulin; ALL, acute lymphocytic leukemia; AML,acute myelogenous leukemia; ATG, antithymocyte globulin; ATR, ataxia-telangiectasia mutated and rad3-related kinase; BFU–E, erythroid burst-formingunits; CD, cluster of differentiation; CFU-GM, colony-forming unit–granulocyte-macrophage; CMV, cytomegalovirus; EBV, Epstein-Barr virus; G, guanine; G-CSF,granulocyte colony-stimulating factor; HHV, human herpes virus; HIV, humanimmunodeficiency virus; HLA, human leukocyte antigen; IL, interleukin; LDH,lactic dehydrogenase; NMRI, nuclear magnetic resonance imaging; PCP,pentachlorophenol; PNH, paroxysmal nocturnal hemoglobinuria; SCF, stemcell factor; T, thymine; TNF, tumor necrosis factor; TNT, trinitrotoluene; TPO,thrombopoietin.

464

PART VI: The Erythrocyte

twofold or greater incidence in the Orient compared to the Occidentmay be multifactorial,

16

but a predisposition gene or genes is a likelycomponent.

12,17

Studies have not established the use of chlorampheni-col in Asia as a cause. Poorly regulated exposure of workers to benzeneis a factor,

18

but the attributable risk from benzene and other toxicexposures does not explain the magnitude of the difference in the inci-dence in Asia compared to that in Europe and South America.

16,17

Arelationship to impure water use in Thailand has led to speculation ofan infectious etiology, although no agent, including seronegative hepa-titis, a known association with the onset of acquired aplastic anemia,

16

has been identified. Seronegative viral hepatitis is a forerunner ofapproximately 7 percent of cases of acquired aplastic anemia.

17

Themale-to-female incidence ratio of aplastic anemia in most studies isapproximately one.

17

■

ETIOLOGY AND PATHOGENESIS

Table 34–2 lists the potential causes for aplastic anemia.The final common pathway to the clinical disease is a decrease in

blood cell formation in the marrow. The number of marrow CD34+cells (multipotential hematopoietic progenitors) and their derivativecolony-forming unit–granulocyte-macrophage (CFU-GM) and burst-forming unit–erythroid (BFU–E) are reduced markedly in patientswith aplastic anemia.

19–22

Long-term culture-initiating cells, an

in vitro

surrogate assay for hematopoietic stem cells, also are reduced toapproximately 1 percent of normal values.

22

Potential mechanismsresponsible for acquired marrow cell failure include (1) direct toxicityto hematopoietic multipotential cells, (2) a defect in the stromalmicroenvironment of the marrow required for hematopoietic celldevelopment, (3) impaired production or release of essential multilin-eage hematopoietic growth factors, (4) cellular or humoral immunesuppression of the marrow multipotential cells, and (5) progressive ero-sion of chromosome telomeres. There is little experimental evidencefor a stromal microenvironmental defect or a deficit of critical hemato-poietic growth factors, and the role of telomerase mutations with con-sequent telomere shortening is unclear, although present in as much as40 percent of patients.

23

Deficiencies in telomere repair could predis-pose to aplastic anemia by affecting the size of the multipotential hema-topoietic cell compartment and by decreasing the multipotential cell’sresponse to a marrow injury, and could play a role in the evolution ofaplastic anemia to a clonal myeloid disease by contributing to genomeinstability.

23

Thus, reduced hematopoiesis in most cases of aplastic ane-mia results from cytotoxic T-cell-mediated immune suppression of

very early CD34+ hematopoietic multipotential progenitor or stemcells.

24

A small fraction of cases is initiated by a toxic exposure, drugexposure, or viral infection, but in these cases the pathogenesis also

TABLE 34–1.

Degree of Severity of Acquired Aplastic Anemia

Diagnostic Categories Hemoglobin

Reticulocyte Concentration

Neutrophil Count

Platelet Count Marrow Biopsy Comments

Moderately severe <100 g/L <40

×

10

9

/L <1.5

×

10

9

/L <50

×

10

9

/L Marked decrease of hemato-poietic cells.

At the time of diagnosis at least 2 of 3 blood counts should meet these criteria.

Severe <90 g/L <30.0

×

10

9

/L <0.5

×

10

9

/L <30.0

×

10

9

/L Marked decrease or absence of hematopoietic cells.

Search for a histocompatible sibling should be made if age permits.

Very Severe <80 g/L <20.0

×

10

9

/L <0.2

×

10

9

/L <20.0

×

10

9

/L Marked decrease or absence of hematopoietic cells.

Search for a histocompatible sibling should be made if age permits.

NOTE

: These values are approximations and must be considered in the context of an individual patient’s situation. (In some clinical trials, the blood count thresholds for moderately severe aplas-tic anemia are higher, e.g., platelet count <100

×

10

9

/L and absolute reticulocyte count <60,000

×

10

9

/L.) The marrow biopsy may contain the usual number of lymphocytes and plasma cells;“hot spots,” focal areas of erythroid cells, may be seen. No fibrosis, abnormal cells, or malignant cells should be evident in the marrow. Dysmorphic features of blood or marrow cells are notfeatures of acquired aplastic anemia. Ethnic differences in the lower limit of the absolute neutrophil count should be considered. (See Chap. 65.)

TABLE 34–2.

Etiologic Classification of Aplastic Anemia

AcquiredAutoimmuneDrugs

See Table 34–3Toxins

BenzeneChlorinated hydrocarbonsOrganophosphates

VirusesEpstein-Barr virusNon-A, -B, -C, -D, -E, or -G hepatitis virusHuman immunodeficiency virus (HIV)

Paroxysmal nocturnal hemoglobinuriaAutoimmune/connective tissue disorders

Eosinophilic fasciitisImmune thyroid disease (Graves disease, Hashimoto thyroiditis)Rheumatoid arthritisSystemic lupus erythematosus

ThymomaPregnancyIatrogenic

RadiationCytotoxic drug therapy

HereditaryFanconi anemiaDyskeratosis congenitaShwachman-Diamond syndromeOther rare syndromes (see Table 34–8)

CHAPTER 34: Aplastic Anemia: Acquired and Inherited

465

may relate to autoimmunity as there is evidence of immune dysfunc-tion in seronegative hepatitis, after benzene exposure, and many suchpatients respond to anti–T-cell therapy.

24

Autoreactive Cytotoxic T Lymphocytes

In vitro

and clinical observations have resulted in the identification of acytotoxic T-cell-mediated attack on multipotential hematopoietic cellsin the CD34+ cellular compartment as the basis for acquired aplasticanemia.

25

Cellular immune injury to the marrow after drug-, viral-, ortoxin-initiated marrow aplasia could result from the induction ofneoantigens that provoke a secondary T-cell-mediated attack on hema-topoietic cells. This mechanism could explain the response to immuno-suppressive treatment in cases that follow exposure to an exogenousagent. Spontaneous or mitogen-induced increases in mononuclear cellproduction of interferon-

γ

,

26,27

interleukin (IL)-2,

27

and tumor necrosisfactor-

α

(TNF-

α

)

28,29

occur. Elevated serum levels of interferon-

γ

arepresent in 30 percent of patients with aplastic anemia, and interferon-

γ

expression has been detected in the marrow of most patients withacquired aplastic anemia.

30

Addition of antibodies to interferon-

γ

enhances

in vitro

colony growth of marrow cells from affectedpatients.

31

Long-term marrow cultures manipulated to elaborate exag-

gerated amounts of interferon-

γ

, markedly reduced the frequency oflong-term culture-initiating cells.

24

These observations indicate thatacquired aplastic anemia is the result of cellular immune-inducedapoptosis of primitive CD34+ multipotential hematopoietic progeni-tors, mediated by cytotoxic T lymphocytes, in part, through the expres-sion of T-helper type 1 (Th1) inhibitory cytokines, interferon-

γ

,

andTNF-

α

(Fig. 34–1).

32

The secretion of interferon-

γ

is a result of theupregulation of transcription regulatory factor T-bet,

33

and apoptosisof CD34+ cells is, in part, mediated through a FAS-dependent path-way.

24

Because HLA-DR2 is more prevalent in patients with aplasticanemia, antigen recognition may be a factor in those patients. A varietyof other potential factors have been found in some patients, includingnucleotide polymorphisms in cytokine genes, overexpression of per-forin in marrow cells, and decreased expression of SAP, a modulatorprotein that inhibits interferon-

γ

secretion.

24

A decrease in regulatory T cells contributes to the expansion of anautoreactive CD8+CD28– T-cell population, which induces apoptosisof autologous hematopoietic multipotential hematopoietic cells.

34

Onemouse model of immune-related marrow failure, induced by infusionof parental lymph node cells into F1 hybrid recipients, caused a fatalaplastic anemia. The aplasia could be prevented by immunotherapy or

FIGURE 34–1.

Immune pathogenesis of apoptosis of CD34 multipotential hematopoietic cells in acquired aplastic anemia. Antigens are presented to T lymphocytes by antigen-presenting cells (APCs). This triggers T cells to activate and proliferate. T-bet, a transcription factor, binds to the interferon-

γ

(IFN-

γ

) promoter region and induces gene expression.SLAM-associated protein (SAP) binds to Fyn and modulates the signaling lymphocyte activation molecule (SLAM) activity on IFN-

γ

expression, diminishing gene transcription. Pa-tients with aplastic anemia show constitutive T-bet expression and low SAP levels. IFN-

γ

and tumor necrosis factor-

α

(TNF-

α

) upregulate both the T cell’s cellular receptors and theFas receptor. Increased production of interleukin-2 leads to polyclonal expansion of T cells. Activation of the Fas receptor by the Fas ligand leads to apoptosis of target cells. Some ef-fects of IFN-

γ

are mediated through interferon regulatory factor 1 (IRF-1), which inhibits the transcription of cellular genes and entry into the cell cycle. IFN-

γ

is a potent inducer ofmany cellular genes, including inducible nitric oxide synthase (NOS), and production of nitric oxide (NO) may diffuse its cytotoxic effects. These events ultimately lead to reducedcell cycling and cell death by apoptosis.

(Reproduced from Young NS, Calado RT, Scheinberg P: Current concepts in the pathophysiology and treatment of aplastic anemia.

Blood

108:2509, 2006, with permission of The American Society of Hematology.)

466


with monoclonal antibodies to interferon-

γ

and TNF-

α

.

24

Anothermouse model of aplastic anemia induced by the infusion of lymph nodecells histoincompatible for the minor H antigen, H60, resulted from theexpansion of H60-specific CD8 T cells in recipient mice. The result wassevere marrow aplasia. The effect of the CD8 T cells could be abrogatedby either immunosuppressive agents or administration of CD4+CD25+regulatory T cells,

35

providing additional experimental evidence for therole of regulatory T cells in the prevention of aplastic anemia.

Several putative target antigens on affected hematopoietic cells havebeen identified. Autoantibodies to one putative antigen, kinectin, havebeen found in patients with aplastic anemia. T cells, responsive tokinectin-derived peptides, suppress granulocyte-monocyte colonygrowth

in vitro

. However, in these studies cytotoxic T lymphocytes withthat specificity were not isolated from patients.

36

Drugs

Chloramphenicol is the most notorious drug documented to causeaplastic anemia. Although this drug is directly myelosuppressive at veryhigh dose because of its effect on mitochondrial DNA, the occurrenceof aplastic anemia appears to be idiosyncratic, perhaps related to aninherited sensitivity to the nitroso-containing toxic intermediates.

37

This sensitivity may produce immunologic marrow suppression, as asubstantial proportion of affected patients respond to treatment withimmunosuppressive therapy.

38

The risk of developing aplastic anemiain patients treated with chloramphenicol is approximately 1 in 20,000,or 25 times that of the general population.

39

Although its use as an anti-biotic has been largely abandoned in industrialized countries, globalreports of fatal aplastic anemia continue to appear with topical or sys-temic use of the drug.

50,51

Epidemiologic evidence established that quinacrine (Atabrine)increased the risk of aplastic anemia.

40

This drug was administered toall U.S. troops in the South Pacific and Asiatic theaters of operations asprophylaxis for malaria during 1943 and 1944. The incidence of aplas-tic anemia was 7 to 28 cases per 1,000,000 personnel per year in theprophylaxis zones, whereas untreated soldiers had 1 to 2 cases per1,000,000 personnel per year. The aplasia occurred during administra-tion of the offending agent and was preceded by a characteristic rash innearly half the cases. Many other drugs have been reported to increasethe risk of aplastic anemia, but owing to incomplete reporting of infor-mation and the infrequency of the association, the spectrum of drug-induced aplastic anemia may not be fully appreciated. Table 34–3 is apartial list of drugs that have been implicated.

41–51

Many of these drugs are known to also induce selective cytopenias,such as agranulocytosis, which usually are reversible after discontinua-tion of the offending agent. These reversible reactions are not correlatedwith the risk of aplastic anemia, casting doubt on the effectiveness of rou-tine monitoring of blood counts as a strategy to avoid aplastic anemia.

Because aplastic anemia is a rare event with drug use, it may occurbecause of an underlying metabolic or immunologic predisposition(gene polymorphism) in susceptible individuals. In the case of phe-nylbutazone-associated marrow aplasia, there is delayed oxidation andclearance of a related compound, acetanilide, as compared to eithernormal controls or those with aplastic anemia from other causes. Thisfinding suggests excess accumulation of the drug as a potential mecha-nism for the aplasia. In some cases, drug interactions or synergy may berequired to induce marrow aplasia. Cimetidine, a histamine H

2

-receptorantagonist, is occasionally implicated in the onset of cytopenias andaplastic anemia, perhaps owing to a direct effect on early hematopoieticprogenitor cells.

52

This drug accentuates the marrow-suppressiveeffects of the chemotherapy drug carmustine.

53

In several instances, ithas been reported as a possible cause of marrow aplasia when givenwith chloramphenicol.

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

Toxic Chemicals

Benzene was the first chemical linked to aplastic anemia, based on stud-ies in factory workers before the 20th century.

54–59

Benzene is used as asolvent and is employed in the manufacture of chemicals, drugs, dyes,and explosives. It has been a vital chemical in the manufacture of rubberand leather goods and has been used widely in the shoe industry, leadingto an increased risk for aplastic anemia (and acute myelogenous leuke-mia) in workers exposed to a poorly regulated environment.

56

In studiesin China, aplastic anemia among workers was sixfold higher than in thegeneral population.

18

The U.S. Occupational Safety and Health Administration has low-ered the permissible atmospheric exposure limit of benzene to 1 partper million (ppm). Previous to that regulatory change, the frequency ofaplastic anemia in workers exposed to greater than 100 ppm benzenewas approximately 1 in 100 workers, which decreased to 1 in 1000workers at 10 to 20 ppm exposure.

55

Organochlorine and organophosphate pesticide compounds havebeen suspected in the onset of aplastic anemia

57,58

and several studieshave indicated an increased relative risk, especially for agriculturalexposures

11,16,59,60

and household

11,60

exposures. These relationshipsare suspect because dose–disease relationships and other importantfactors have not been delineated, and several studies have not found anassociation with environmental exposures.

12,61

DDT (dichlorodiphenyl-trichloroethane), lindane, and chlordane are insecticides that have alsobeen associated with cases of aplastic anemia.

16,58

Occasional cases stilloccur following heavy exposure at industrial plants or after its use as apesticide.

62

Lindane is metabolized in part to pentachlorophenol(PCP), another potentially toxic chlorinated hydrocarbon that is manu-factured for use as a wood preservative. Many cases of aplastic anemiaand related blood disorders have been attributed to PCP over the past25 years.

58,63

Prolonged exposures to petroleum distillates in the formof Stoddard solvent

64

and acute exposure to toluene through the prac-tice of glue sniffing

65,66

also have been reported to cause marrow apla-sia. Trinitrotoluene (TNT), an explosive used extensively during WorldWars I and II, is absorbed readily by inhalation and through the skin.

67

Fatal cases of aplastic anemia were observed in munitions workersexposed to TNT in Great Britain

68

from 1940 to 1946. In most cases,these conclusions have not been derived from specific studies but fromaccumulation of case reports or from patient histories, making conclu-sions provisional, although the argument for minimizing exposures topotential toxins is logical in any case.

Viruses

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

A relationship between hepa-titis and the subsequent development of aplastic anemia has been thesubject of a number of case reports, and this association was empha-sized by two major reviews in the 1970s.

69,70

In the aggregate, thesereports summarized findings in more than 200 cases. In manyinstances, the hepatitis was improving or had resolved when theaplastic anemia was noted 4 to 12 weeks later. Approximately 10 per-cent of cases occurred more than 1 year after the initial diagnosis ofhepatitis. Most patients were young (ages 18 to 20 years); two-thirdswere male, and their survival was short (10 weeks). Although hepati-tis A and B have been implicated in aplastic anemia in a small num-ber of cases, most cases are related to non-A, non-B, non-Chepatitis.

71–73

Severe aplastic anemia developed in 9 of 31 patientswho underwent liver transplantation for non-A, non-B, non-C hepatitis,

CHAPTER 34: Aplastic Anemia: Acquired and Inherited

467

but in none of 1463 patients transplanted for other indications.

75

Sev-eral lines of evidence indicate there is no causal association withhepatitis C virus, suggesting that an unknown viral agent isinvolved.

16,75,76

Hepatitis virus B or C can be a secondary infection, ifcarefully screened blood products are not used for transfusion. In 15patients with posthepatitic aplastic anemia, no evidence was foundfor hepatitis A, B, C, D, E, or G, transfusion-transmitted virus, orparvovirus B19.

76

Several reports suggest a relationship of parvovirusB19 to aplastic anemia,

77,78

whereas others have not.

79

This relation-ship has not been established (see Chap. 35). The effect of seronega-tive hepatitis may be mediated through an autoimmune T-cell effectbecause of evidence of T-cell activation and cytokine elaboration.

24

These patients also have a similar response to combined immuno-

therapy as does idiopathic aplastic anemia (see “TREATMENT,Combination Immunotherapy”).

Epstein-Barr Virus

Epstein-Barr virus (EBV) has been implicated in thepathogenesis of aplastic anemia.

80,81

The onset usually occurs within 4 to6 weeks of infection. In some cases, infectious mononucleosis is subclini-cal, with a finding of reactive lymphocytes in the blood film and serolog-ical results consistent with a recent infection (see Chap. 84). EBV hasbeen detected in marrow cells,

81

but it is uncertain whether marrow apla-sia results from a direct effect or an immunologic response by the host.Patients have recovered following therapy with antithymocyte globulin.

81

Other Viruses

Human immunodeficiency virus (HIV) infection fre-quently is associated with varying degrees of cytopenia. The marrow is

TABLE 34–3.

Drugs Associated with Aplastic Anemia

Category High Risk Intermediate Risk Low Risk

Analgesic Phenacetin, aspirin, salicylamide

Antiarrhythmic Quinidine, tocainide

Antiarthritic Gold salts Colchicine

Anticonvulsant Carbamazepine, hydantoins, felbamate

Ethosuximide, phenacemide, primidone, trimethadione, sodium valproate

Antihistamine Chlorpheniramine, pyrilamine, tripelennamine

Antihypertensive Captopril, methyldopa

Antiinflammatory Penicillamine, phenylbutazone, oxyphenbutazone

Diclofenac, ibuprofen, indomethacin, naproxen, sulindac

Antimicrobial

Antibacterial Chloramphenicol Dapsone, methicillin, penicillin, streptomycin,

β

-lactam antibiotics

Antifungal Amphotericin, flucytosine

Antiprotozoal Quinacrine Chloroquine, mepacrine, pyrimethamine

Antineoplastic drugs

Alkylating agent Busulfan, cyclophosphamide, melphalan, nitrogen mustard

Antimetabolite Fluorouracil, mercaptopurine, methotrexate

Cytotoxic antibiotic Daunorubicin, doxorubicin, mitoxantrone

Antiplatelet Ticlopidine

Antithyroid Carbimazole, methimazole, methylthiouracil, potassium perchlorate, propylthiouracil, sodium thiocyanate

Sedative and tranquilizer Chlordiazepoxide, chlorpromazine (and other phenothiazines), lithium, meprobamate, methyprylon

Sulfa derivative Sulfonamides

Antibacterial Numerous sulfonamides

Diuretic Acetazolamide Chlorothiazide, furosemide

Hypoglycemic Chlorpropamide, tolbutamide

Miscellaneous Allopurinol, interferon, pentoxifylline, penicillamine

NOTE

: Drugs that invariably cause marrow aplasia with high doses are termed

high risk;

drugs with 30 or more reported cases are listed as moderate risk; others are less often associated withaplastic anemia (low risk).

SOURCE

: This list was compiled from the AMA Registry,

41

publications of the International Agranulocytosis and Aplastic Anemia Study,

42–46

other reviews and studies,

24,47–50

previous compila-tions of offending agents,

51

and selected reports. An additional comprehensive source for potentially offending drugs can be found in

The Drug Etiology of Agranulocytosis and Aplastic Ane-mia,

Oxford, UK:

Oxford University Press, 1991.

468


often cellular, but occasional cases of aplastic anemia have beennoted.

82–84

In these patients, marrow hypoplasia may result from viralsuppression and from the drugs used to control viral replication in thisdisorder. Human herpes virus (HHV)-6 has caused severe marrowaplasia subsequent to marrow transplantation for other disorders.85

Autoimmune DiseasesThe incidence of severe aplastic anemia was sevenfold greater thanexpected in patients with rheumatoid arthritis.47 It is uncertain whetherthe aplastic anemia is related directly to rheumatoid arthritis or to thevarious drugs used to treat the condition (gold salts, D-penicillamine,and nonsteroidal antiinflammatory agents). Occasional cases of aplasticanemia are seen in conjunction with systemic lupus erythematosus.86

In vitro studies found either the presence of an antibody87 or suppressorcell88,89 directed against hematopoietic progenitor cells. Patients haverecovered after plasmapheresis,87 glucocorticoids,89 or cyclophospha-mide therapy,88,90 which is compatible with an immune etiology.

Eosinophilic fasciitis, an uncommon connective tissue disorder withpainful swelling and induration of the skin and subcutaneous tissue, hasbeen associated with aplastic anemia.91,92 Although it may be antibody-mediated in some cases, it has been largely unresponsive to therapy.91

Nevertheless, (1) stem cell transplantation, (2) immunosuppressive ther-apy using cyclosporine,91 (3) immunosuppressive therapy using anti-thymocyte globulin (ATG), or (4) immunosuppressive therapy withATG and cyclosporine cures or significantly ameliorates the diseasein a few patients.92

Severe aplastic anemia also has been reported coincident withimmune thyroid disease (Graves disease)93–97 and the aplasia has beenreversed with treatment of the hyperthyroidism. Aplastic anemia hasoccurred in association with thymoma.98–103 Autoimmune renal dis-ease and aplastic anemia have occurred concurrently. The underlyingrelationship may be the role of cytotoxic T lymphocytes in the patho-genesis of several autoimmune diseases and in aplastic anemia.104

PregnancyThere are a number of reports of pregnancy-associated aplastic anemia,but the relationship between the two conditions is not always clear.105–110

In some patients, preexisting aplastic anemia is exacerbated with preg-nancy, only to improve following termination of the pregnancy.105,106 Inother cases, the aplasia develops during pregnancy with recurrencesduring subsequent pregnancies.106,107 Termination of pregnancy ordelivery may improve the marrow function, but the disease mayprogress to a fatal outcome even after delivery.105–107 Therapy mayinclude elective termination of early pregnancy, supportive care, immu-nosuppressive therapy, or marrow transplantation after delivery. Preg-nancy in women previously treated with immunosuppression foraplastic anemia can result in the birth of a normal newborn.110 In thislatter study of 36 pregnancies, 22 were uncomplicated, 7 were compli-cated by a relapse of the marrow aplasia, and 5 without marrow aplasiarequired red cell transfusion during delivery.110 One death occurredfrom cerebral thrombosis in a patient with paroxysmal nocturnalhemoglobinuria (PNH) and marrow aplasia.

Iatrogenic CausesAlthough marrow toxicity from cytotoxic chemotherapy or radiationproduces direct damage to stem cells and more mature cells, resultingin marrow aplasia, most patients with acquired aplastic anemia cannotrelate an exposure that would be responsible for marrow damage.

Chronic exposure to low doses of radiation or use of spinal radiationfor ankylosing spondylitis is associated with an increased, but delayed,risk of developing aplastic anemia and acute leukemia.111,112 Patients

who were given thorium dioxide (Thorotrast) as an intravenous con-trast medium suffered numerous late complications, including malig-nant liver tumors, acute leukemia, and aplastic anemia.113 Chronicradium poisoning with osteitis of the jaw, osteogenic sarcoma, andaplastic anemia was seen in workers who painted watch dials withluminous paint when they moistened the brushes orally.114

Acute exposures to large doses of radiation are associated with thedevelopment of marrow aplasia and a gastrointestinal syndrome.115,116

Total body exposure to between 1 and 2.5 Gy leads to gastrointestinalsymptoms and depression of leukocyte counts, but most patientsrecover. A dose of 4.5 Gy leads to death in half the individuals (LD50)owing to marrow failure. Higher doses in the range of 10 Gy are univer-sally fatal unless the patient receives extensive supportive care followedby marrow transplantation. Aplastic anemia associated with nuclearaccidents was seen after the disaster that occurred at the Chernobylnuclear power station in the Ukraine in 1986.117

Antineoplastic drugs such as alkylating agents, antimetabolites, andcertain cytotoxic antibiotics have the potential for producing marrowaplasia. In general, this is transient, is an extension of their pharmaco-logic action, and resolves within several weeks of completing chemo-therapy. Although unusual, severe marrow aplasia can follow use of thealkylating agent, busulfan, and may persist indefinitely. Patients maydevelop marrow aplasia 2 to 5 years after discontinuation of alkylatingagent therapy. These cases often evolve into hypoplastic myelodysplas-tic syndromes.

Stromal Microenvironment and Growth FactorsShort-term clonal assays for marrow stromal cells have shown variabledefects in stromal cell function. Serum levels of stem cell factor (SCF)have been either moderately low or normal in several studies of aplasticanemia.118,119 Although SCF augments the growth of hematopoieticcolonies from aplastic anemia patient’s marrows, its use in patients hasnot led to clinical remissions. Another early acting growth factor, FLT-3ligand, is 30- to 100-fold elevated in the serum of patients with aplasticanemia.120 Fibroblasts grown from patients with severe aplastic anemiahave subnormal cytokine production. However, serum levels of granu-locyte colony-stimulating factor,121 erythropoietin,122 and thrombopoi-etin (TPO)123 are usually high. Synthesis of IL-1, an early stimulator ofhematopoiesis, is decreased in mononuclear cells from patients withaplastic anemia.124 Studies of the microenvironment have shown rela-tively normal stromal cell proliferation and growth factor produc-tion.125 These findings, coupled with the limited response of patientswith aplastic anemia to growth factors, suggest that cytokine deficiencyis not the etiologic problem in most cases. The most compelling argu-ment is that most patients transplanted for aplastic anemia are curedwith allogeneic donor stem cells and autologous stroma.126

A rare exception is the homozygous or mixed heterozygous mutation ofthe TPO receptor gene, MPL, which can cause amegakaryocytic thrombo-cytopenia that evolves, later, into aplastic anemia (see Chap. 119).

■ CLINICAL FEATURESThe onset of symptoms of aplastic anemia may be gradual with pallor,weakness, dyspnea, and fatigue as a result of the anemia. Dependent pete-chiae, bruising, epistaxis, vaginal bleeding, and unexpected bleeding atother sites secondary to thrombocytopenia are frequent presenting signs ofthe underlying marrow disorder. Rarely, it may be more dramatic withfever, chills, and pharyngitis or other sites of infection resulting from neu-tropenia and monocytopenia. Physical examination generally is unreveal-ing except for evidence of anemia (e.g., conjunctival and cutaneous pallor,resting tachycardia) or cutaneous bleeding (e.g., ecchymoses and pete-chiae), gingival bleeding and intraoral purpura. Lymphadenopathy and

CHAPTER 34: Aplastic Anemia: Acquired and Inherited 469

splenomegaly are not features of aplastic anemia; such findings suggest analternative diagnosis such as a clonal myeloid or lymphoid disease.

■ LABORATORY FEATURESBlood FindingsPatients with aplastic anemia have varying degrees of pancytopenia.Anemia is associated with a low reticulocyte index. The relative reticu-locyte count is usually less than 1 percent and may be zero despite thehigh levels of erythropoietin. Absolute reticulocyte counts are usuallyfewer than 40,000/μL (40 × 109/L). Macrocytes may be present. Theabsolute neutrophil and monocyte count are low. An absolute neutro-phil count fewer than 500/μL (0.5 × 109/L) along with a platelet countfewer than 30,000/μL (30 × 109/L) is indicative of severe disease and a

neutrophil count below 200/μL (0.2 × 109/L) denotes very severe dis-ease (see Table 34–1). Lymphocyte production is thought to be normal,but patients may have mild lymphopenia. Platelets function normally.Significant qualitative changes of red cell, leukocyte, or platelet mor-phology are not features of classical acquired aplastic anemia. On occa-sion, only one cell line is depressed initially, which may lead to an earlydiagnosis of red cell aplasia or amegakaryocytic thrombocytopenia. Insuch patients, other cell lines will fail shortly thereafter (days to weeks)and permit a definitive diagnosis. Table 34–4 is a plan for initial labora-tory investigation.

Plasma FindingsThe plasma contains high levels of hematopoietic growth factors, includ-ing erythropoietin, thrombopoietin, and myeloid colony-stimulating fac-tors. Plasma iron values are usually high, and 59Fe clearance isprolonged, with decreased incorporation into red cells.

Marrow Findings

Morphology The marrow aspirate typically contains numerous spiculeswith empty, fat-filled spaces, and relatively few hematopoietic cells.Lymphocytes, plasma cells, macrophages, and mast cells may bepresent. On occasion, some spicules are cellular or even hypercellular(“hot spots”), but megakaryocytes usually are reduced. These focalareas of residual hematopoiesis do not appear to be of prognostic sig-nificance. Residual granulocytic cells generally appear normal, but it isnot unusual to see mild macronormoblastic erythropoiesis, presumablyas a result of the high levels of erythropoietin. Marrow biopsy is essen-tial to confirm the overall hypocellularity (Fig. 34–2), as a poor yield ofspicules and cells occurs in marrow aspirates in other disorders, espe-cially if fibrosis is present.

In severe aplastic anemia, as defined by the International AplasticAnemia Study Group, less than 25 percent cellularity or less than 50percent cellularity with less than 30 percent hematopoietic cells is seenin the marrow.Progenitor Cell Growth In vitro CFU-GM and BFU–E colony assaysreveal a marked reduction in progenitor cells.19–22

Cytogenetic Studies Cytogenetic analysis may be difficult to performowing to low cellularity; thus, multiple aspirates may be required to

TABLE 34–4. Approach to Diagnosis

History and Physical Examination

• Complete blood counts, reticulocyte count, and examination of the blood film• Marrow aspiration and biopsy• Marrow cell cytogenetics to evaluate clonal myeloid disease• Fetal hemoglobin level and DNA stability test as markers of Fanconi anemia• Immunophenotyping of red and white cells, especially for CD55, CD59 to

exclude PNH• Direct and indirect Coombs test to rule out immune cytopenia• Serum lactate dehydrogenase (LDH) and uric acid that if increased may

reflect neoplastic cell turnover• Liver function tests to assess evidence of any recent hepatitis virus exposure• Screening tests for hepatitis viruses A, B, and C• Screening tests for EBV, cytomegalovirus (CMV), and HIV• Serum B12 and red cell folic acid levels to rule out megaloblastic

pancytopenia• Serum iron, iron-binding capacity, and ferritin as a baseline prior to chronic

transfusion therapy

FIGURE 34–2. Marrow biopsy in aplastic anemia. A. A normal marrow biopsy section of a young adult. B. The marrow biopsy section of a young adult with very severe aplasticanemia. The specimen is devoid of hematopoietic cells and contains only scattered lymphocytes and stromal cells. The hematopoietic space is replaced by reticular cells (pre-adipocyticfibroblasts) converted to adipocytes.

A B

470 PART VI: The Erythrocyte

provide sufficient cells for study. The results are normal in aplasticanemia. Clonal cytogenetic abnormalities in otherwise apparent aplas-tic anemia is indicative of an underlying hypoaccumulative clonalmyeloid disease.127

Imaging Studies Magnetic resonance imaging can be used to distin-guish between marrow fat and hematopoietic cells.128 This may be amore useful overall estimate of marrow hematopoietic cell density thanmorphologic techniques and may help differentiate hypoplasticmyelogenous leukemia from aplastic anemia.128

■ DIFFERENTIAL DIAGNOSISAny disease that can present with pancytopenia may mimic aplasticanemia if only the blood counts are considered. Measurement of thereticulocyte count and an examination of the blood film and marrowbiopsy are essential early steps to arrive at a diagnosis. A reticulocytepercentage of 0.5 percent to zero is strongly indicative of aplastic eryth-ropoiesis, and when coupled with leukopenia and thrombocytopenia,points to aplastic anemia. Absence of qualitative abnormalities of cellson the blood film and a markedly hypocellular marrow are characteris-tic of acquired aplastic anemia. The disorders most commonly con-fused with severe aplastic anemia include the approximately 5 to 10percent of patients with myelodysplastic syndromes who present with ahypoplastic rather than a hypercellular marrow. Myelodysplasia shouldbe considered if there is abnormal blood film morphology consistentwith myelodysplasia (e.g., poikilocytosis, basophilic stippling, neutro-phils with the pseudo-Pelger-Hüet anomaly). Marrow erythroid pre-cursors in myelodysplasia may have dysmorphic features. Pathologicsideroblasts are inconsistent with aplastic anemia and a frequent featureof myelodysplasia. Granulocyte precursors may have reduced or abnor-mal granulation. Megakaryocytes may have abnormal nuclear lobula-tion (e.g., unilobular micromegakaryocytes; see Chap. 88). If clonalcytogenetic abnormalities are found, a clonal myeloid disorder, espe-cially myelodysplastic syndrome or hypocellular myelogenous leuke-mia is likely. Magnetic resonance imaging (MRI) studies of bone maybe useful in differentiating severe aplastic anemia from clonal myeloidsyndromes. The former gives a fatty signal and the latter a diffuse cellu-lar pattern.

A hypocellular marrow frequently is associated with PNH. PNH ischaracterized by an acquired mutation in the PIG-A gene that encodesan enzyme that is required to synthesize mannolipids. The latter defi-ciency prevents the synthesis of the glycosyl-phosphatidylinositolanchor precursor. This moiety anchors several proteins, includinginhibitors of the complement pathway to blood cell membranes, and itsabsence accounts for the complement-mediated hemolysis in PNH. Asmany as 50 percent of patients with otherwise typical aplastic anemiahave evidence of glycosyl-phosphatidylinositol molecule defects anddiminished phosphatidylinositol-anchored protein on leukocytes andred cells as judged by flow cytometry, analogous to that seen in PNH.129

The decrease or absence of these membrane proteins may make thePNH clone of cells resistant to the acquired immune attack on normalmarrow components, or the phosphatidylinositol-anchored protein(s)on normal cells provides an epitope that initiates an aberrant T-cellattack, leaving the PNH clone relatively resistant (see Chap. 40).24

Occasionally, apparent aplastic anemia may be the prodrome tochildhood130 or, less commonly, adult131 acute lymphoblastic leukemia.Sometimes, careful examination of marrow cells by light microscopy orflow cytometry will uncover a population of leukemic lymphoblasts. Inother cases, the acute leukemia may appear later. Hairy-cell leukemia,Hodgkin disease, or another lymphoma subtype, rarely, may be pre-ceded by a period of marrow hypoplasia. Immunophenotyping of mar-row and blood cells by flow cytometry for CD25 may uncover the

presence of hairy cells. Other clinical features may be distinctive (seeChap. 95). Organomegaly such as lymphadenopathy, hepatomegaly, orsplenomegaly are inconsistent with the atrophic (hypoproliferative) fea-tures of aplastic anemia. Large granular lymphocytic leukemia has alsobeen associated with aplastic anemia. Rare cases of typical acquiredaplastic anemia have been followed by t(9;22)-positive acute lympho-cytic leukemia (ALL), or chronic myelogenous leukemia (CML).131

■ RELATIONSHIP AMONG APLASTIC ANEMIA, PNH, AND CLONAL MYELOID DISEASES

In addition to the diagnostic difficulties occasionally presented bypatients with hypoplastic myelodysplastic syndromes, hypoplasticacute myelogenous leukemia (AML), or PNH with hypocellular mar-rows, there may be a more fundamental relationship among these threediseases and aplastic anemia. The development of clonal cytogeneticabnormalities such as monosomy 7 or trisomy 8 in a patient with aplas-tic anemia portends the evolution of a myelodysplastic syndrome oracute leukemia. Occasionally, these cytogenetic markers have beentransient, and in cases with disappearance of monosomy 7, hematologicimprovement has occurred as well.132 Persistent monosomy 7 carries apoor prognosis as compared to trisomy 8.133,134

As many as 15 to 20 percent of patients with aplastic anemia have a5-year probability of developing myelodysplasia.132 If one excludesany transformation to a clonal myeloid disorder that occurs up to 6months after treatment to avoid misdiagnosis among the hypoplasticclonal myeloid diseases, the frequency of a clonal disorder was nearly15 times greater in patients treated with immunosuppression as com-pared to those treated with marrow transplantation after 39 months ofobservation.135 This finding suggests either that immune suppressionby anti–T-cell therapy enhances the evolution of a neoplastic clone orthat it does not suppress the intrinsic tendency of aplastic anemia toevolve to a clonal disease, but provides the increased longevity of thepatient required to express that potential. The latter interpretation ismore likely as patients successfully treated solely with androgensdevelop clonal disease as frequently as those treated with immuno-suppression.136 Transplantation may reduce the potential to clonalevolution in patients with aplastic anemia by reestablishing robustlymphohematopoiesis.

Telomere shortening also may play a pathogenetic role in the evolutionof aplastic anemia into myelodysplasia. Patients with aplastic anemiahave shorter telomere lengths than matched controls, and patients withaplastic anemia with persistent cytopenias had greater telomere shorten-ing over time than matched controls. Three of five patients with telomerelengths less than 5 kb developed clonal cytogenetic changes, whereaspatients with longer telomeres did not develop such diseases.23,137

The relationship of PNH to aplastic anemia remains enigmatic.Because hematopoietic stem cells lacking the phosphatidylinositol-anchored proteins are present in many or all normal persons in verysmall numbers,138 it is not surprising that more than 50 percent ofpatients with aplastic anemia may have a PNH cell population asdetected by immunophenotyping.129 The probability of patients withaplastic anemia developing a clinical syndrome consistent with PNH is10 to 20 percent, and this is not a consequence of immunosuppressivetreatment.132 Patients also may present with the hemolytic anemia ofPNH and later develop progressive marrow failure so that any pathoge-netic explanation should consider both types of development of aplasticmarrows in PNH. The PIG-A mutation may confer either a proliferativeor survival advantage to PNH cells.139 A survival advantage could resultif the anchor protein or one of its ligands served as an epitope for the T-lymphocyte cytotoxicity inducing the marrow aplasia. In this case, thepresenting event could either reflect cytopenias or the sensitivity of red


cells to complement lysis and hemolysis, depending on the intrinsicproliferative potential of the PNH clone.

Within our current state of knowledge, aplastic anemia is an autoim-mune process, and any residual hematopoiesis is presumably polyclo-nal. This is a critical distinction from hypoplastic leukemia and PNH,which are clonal (neoplastic) diseases. The environment of the aplasticmarrow, however, may favor the eventual evolution of a mutant (malig-nant) clone, especially if immunotherapy is used, whereas hematopoi-etic stem cell transplantation may either ablate threatening minorclones or establish more robust hematopoiesis, an environment lessconducive to clonal evolution.

■ TREATMENTApproach to TherapySevere anemia, bleeding from thrombocytopenia, and, rarely at the timeof diagnosis, infection secondary to granulocytopenia and monocytope-nia require prompt attention to remove potential life-threatening con-ditions and improve patient comfort (Table 34–5). More specifictreatment of the marrow aplasia involves two principal options: (1) syn-geneic or allogeneic hematopoietic stem cell transplantation or (2) com-bination immunosuppressive therapy with ATG and cyclosporine. Theselection of the specific mode of treatment depends on several factors,including the patient’s age and condition and the availability of a suitableallele-level HLA-matched hematopoietic stem cell donor. In general,transplantation is the preferred treatment for children and most other-wise healthy younger adults. Early histocompatibility testing of siblingsis of particular importance because it establishes whether there is anoptimal donor available to the patient for transplantation. The preferredstem cell source is a histocompatible sibling matched at the HLA-A, B,C, and DR loci.

Supportive Care

The Use of Blood Products Although it was recommended that redcell and platelet transfusions be used sparingly in potential transplantrecipients to minimize sensitization to histocompatibility antigens,this has become less important since ATG and cyclophosphamidehave been used as the preparative regimen for transplantation in

aplastic anemia, as their use has markedly reduced the problem ofgraft rejection.140

Cytomegalovirus (CMV)-reduced risk red cells and platelets shouldbe given to a potential transplant recipient to minimize problems withCMV infections after transplantation. Once a patient is shown to beCMV-positive, this restriction is no longer necessary. Leukocyte-deple-tion filters or CMV serotesting are equivalent methods of decreasingthe risk of transmitting CMV.Red Cell Transfusion Packed red cells to alleviate symptoms of anemiausually are indicated at hemoglobin values below 8 g/dL (80 g/L),unless comorbid medical conditions require a higher hemoglobin con-centration. These products should be leukocyte-depleted to lessen leu-kocyte and platelet sensitization and to reduce subsequent transfusionreactions and radiated to reduce the potential for a graft-versus-hostreaction. It is important not to transfuse patients with red cells (orplatelets) from family members if transplantation within the family isremotely possible, as this approach may sensitize patients to minor his-tocompatibility antigens, increasing the risk of graft rejection aftermarrow transplantation. Following a marrow transplant, or in thoseindividuals in whom transplantation is not a consideration, familymembers may be ideal donors for platelet products. Because each unitof red cells adds approximately 200 to 250 mg of iron to the total bodyiron, over the long-term transfusion-induced iron overload may occur.This is not a major problem in patients who respond to transplantationor immunosuppressive therapy, but it is an issue in nonresponders whorequire continued transfusion support. In the latter case, considerationshould be given to iron chelation therapy. Newer oral agents will makethis procedure easier to effect (see Chap. 47).141

Platelet Transfusion It is important to assess the risk of bleeding in eachpatient. Most patients tolerate platelet counts of 10,000/μL (10 × 109/L)without undue bruising or bleeding, unless a systemic infection ispresent.142,143 A traumatic injury or surgery requires transfusion to>50,000/μL or >100,000/μL, respectively. Administration of ε-ami-nocaproic acid, 100 mg/kg per dose every 4 hours (maximum dose 5 g)orally or intravenously, may reduce the bleeding tendency.144 Pooledrandom-donor platelets may be used until sensitization ensues,although it is preferable to use single-donor platelets from the onset tominimize sensitization to HLA or platelet antigens. Subsequently,single-donor apheresis products or HLA-matched platelets may berequired.

Platelet refractoriness is a major problem with long-term transfusionsupport.145 This may occur transiently, with fever or infection, or as achronic problem secondary to HLA sensitization. In the past, thisoccurred in approximately 50 percent of patients after 8 to 10 weeks oftransfusion support. Filtration of blood and platelet concentrates toremove leukocytes reduces this problem to approximately 15 percent ofpatients receiving chronic transfusions.145,146 Patient’s should also getABO-identical platelets because this enhances platelet survival and fur-ther decreases refractoriness to platelet transfusion. Single-donor HLA-matched apheresis-harvested platelets may be necessary in previouslypregnant or transfused patients who are already allosensitized or whobecome so after treatment with leukoreduced platelets. The frequencyof either of these events is less than 10 percent. Approaches to chronicplatelet transfusion are discussed in Chap. 141. The utility of TPOreceptor agonists in the thrombocytopenia of aplastic anemia is yet tobe determined.Management of Neutropenia Neutropenic precautions should be appliedto hospitalized patients with a severe depression of the neutrophil count.The level of neutrophils requiring precautions is fewer than 500/μL(0.5 × 109/L). One approach is to use private rooms, with requirementsfor face masks and handwashing with antiseptic soap. Unwashed fresh

TABLE 34–5. Initial Management of Aplastic Anemia

• Discontinue any potential offending drug and use an alternative class ofagents if essential.

• Anemia: transfusion of leukocyte-depleted, irradiated red cells as requiredfor very severe anemia.

• Very severe thrombocytopenia or thrombocytopenic bleeding: consider ε-aminocaproic acid; transfusion of platelets as required.

• Severe neutropenia; use infection precautions.• Fever (suspected infection): microbial cultures; broad-spectrum antibiotics

if specific organism not identified, granulocyte colony-stimulating factor (G-CSF) in dire cases. If child or small adult with profound infection (e.g., gram-negative bacteria, fungus, persistent positive blood cultures) canconsider neutrophil transfusion from a G-CSF pretreated donor.

• Immediate assessment for allogeneic stem cell transplantation: Histocom-patibility testing of patient, parents, and siblings. Search databases for unre-lated donor, if appropriate.


fruits and vegetables should be avoided as they aresources of bacterial contamination. It is uncom-mon for patients with aplastic anemia to presentwith a significant infection. When patients withaplastic anemia become febrile, cultures should beobtained from the throat, sputum (if any), blood,urine, stool, and any suspicious lesions. Broad-spectrum bacteriocidal antibiotics should be initi-ated promptly, without awaiting culture results.The choice of antibiotics depends on the preva-lence of organisms and their antibiotic sensitivityin the local setting. Organisms of concern usuallyinclude Staphylococcus aureus (notably methicillin-and oxacillin-resistant strains), Staphylococcus epi-dermidis (in patients with venous access devices),and gram-negative organisms. Patients with persis-tent culture-negative fevers should be consideredfor antifungal treatment (see Chap. 22).

In the past, leukocyte transfusions were used on adaily basis to reduce the short-term mortality frominfections. It was unusual to detect more than 100 to200 neutrophils per microliter for more than a fewhours after transfusion. The yield of neutrophils canbe increased by administering granulocyte colony-stimulating factor (G-CSF) to the donor,147 but mostphysicians avoid using white cell products becausepresent-day antibiotics are usually sufficient to treata patient for an episode of sepsis. Notable exceptionsinclude documented invasive aspergillosis unre-sponsive to amphotericin (particularly in the posttransplant setting),infections with organisms resistant to all known antibiotics, and if bloodcultures remain positive in spite of antibiotic treatment. Leukocyte trans-fusion is more effective in children and adults with smaller body size, astransfused leukocytes have a smaller distribution space, which results inhigher blood and tissue concentrations.Hematopoietic Stem Cell Transplantation Prompt therapy usually isindicated for patients with severe aplastic anemia. The major curativeapproach is hematopoietic stem cell transplantation from a histocom-patible sibling.148–150 This treatment modality is described in Chap. 21.Only 20 to 30 percent of patients in the United States have compatiblesibling donors (related to average family size). In the unusual case of anidentical twin donor, conditioning is required to obliterate the immunedisease in the recipient, but it can be limited to cyclophosphamide. Inthis setting, an 80 to 90 percent survival is expected. Marrow stem cellsseem to perform better than blood stem cells when used as a source forpatients with aplastic anemia, although this is under continued study.The results of transplantation are best in patients younger than age 20years (80 to 90 percent long-term survival) but decrease every decadeof age thereafter. Posttransplant mortality is increased and survivaldecreased with increasing age (Fig. 34–3). In patients older than age 40years, survival in matched sibling transplant is reduced to approxi-mately 50 percent.151 There are still uncertainties about the optimalconditioning program in younger and older patients. ATG, cyclophos-phamide, total-body radiation, and fludarabine are among the agentsbeing studied.148,150,151 The longer the delay between diagnosis andtransplant, the less salutary the outcome, probably as a result of agreater number of transfusions and a higher likelihood of pretransplantinfection. Acute and chronic graft-versus-host disease are serious com-plications, and therapy to prevent or ameliorate them is a standard partof posttransplant treatment.148,151 Transplants have been performedusing stem cells from partially matched siblings or unrelated, histo-compatible donors recruited through the National Marrow Donor Pro-

gram or similar organizations in other countries.152 Umbilical cordblood is an alternative source of stem cells from unrelated donors (or,rarely, siblings) for transplantation in children. The use of high-resolution,HLA typing of a matched, unrelated donor markedly improves theprognosis for transplantation.153 High-resolution DNA matching atHLA-A, -B, -C, and -DRB1 (8 of 8 allele) is considered the lowest levelof matching consistent with the highest level of survival. If there is anHLA mismatch at one or more loci, especially HLA-A or -DRB1, theoutcome is compromised,153 and immunosuppression with combinedtherapy may be preferred initially, depending on patient age, cytomega-lovirus status, and disease severity. The use of hematopoietic stem celltransplantation can be considered for patients who do not respond orwho no longer respond to immunotherapy.151 If the patient in questionis a candidate for stem cell transplantation based on all relevant factors,transplantation could be considered at any age for a patient with a syn-geneic donor; transplantation could be considered as a first-choicetherapy up to age 50 years for a patient with an HLA allele-levelmatched sibling donor; and transplantation could be considered a first-choice therapy if an allele-level HLA-matched unrelated donor is avail-able for patients younger than age 20 years.151

Components of Anti–T-Lymphocyte (Immunosuppressive) TherapyAntilymphocyte Serum and Antithymocyte Globulin ATG and ALG actprincipally by reducing cytotoxic T cells. This involves ATG-inducedapoptosis through both FAS and TNF pathways.154 Cathepsin B alsoplays a role in T-cell cytotoxicity at clinical concentrations of ATG, butmay involve an independent apoptosis pathway.155 ATG and ALG alsorelease hematopoietic growth factors from T cells.156,157 Horse and rab-bit ATG are licensed in the United States. Skin tests against horse serumshould be performed prior to administration.158 If positive, the patientmay be desensitized. ATG therapy is given daily for 4 to 10 days withdoses of 15 to 40 mg/kg. Fever and chills are common during the firstday of treatment. Concomitant treatment with glucocorticoids, such as

FIGURE 34–3. Probability of survival after hematopoietic stem cell transplantation for severe aplastic anemia by do-nor type and age, 1998–2004. Patients receiving marrow from a matched sibling had better outcomes if they wereequal to or less than 20 years of age, compared to those older than 20 years of age. Patients receiving marrow froma matched sibling fared better than those who received marrow from a matched unrelated donor, at any age. Patientsyounger than 20 or older than 20 years of age did not have a significant difference in outcome if they received mar-row from an unrelated matched donor. Id, identical; sib, sibling. (Figure reproduced from the Center for Interna-tional Transplant Research data on the National Marrow Donor Program website. http://www.marrow.org/PATIENT/Undrstnd_Disease_Treat/Lrn_about_Disease/Aplastic_Anemia/AA_Tx_Outcomes/index.html. Last ac-cessed February 2009.)

0

100

90

80

70

60

50

Per

cent

sur

viva

l

HLA-id sib, 20y (N = 915)

HLA-id sib, >20y (N = 822)

Unrelated, 20y (N = 388)

Unrelated, >20y (N = 212)

P < 0.001

1 2 3

Years

4 5 6

40

30

20

10

0

100

90

80

70

60

50

40

30

20

10

0


methylprednisolone or dexamethasone lessens the reaction to ATG.Studies are under way to compare equine to rabbit ATG in the immu-notherapy of aplastic anemia.

ATG treatment may accelerate platelet destruction, reduce the abso-lute neutrophil count, and cause a positive direct antiglobulin test. Thiseffect may lead to an increase in transfusion requirements during the 4-to 10-day treatment interval. Serum sickness, characterized by spikingfevers, skin rashes, and arthralgias, occurs commonly 7 to 10 days fromthe first dose. The clinical manifestations of serum sickness can bediminished by increasing the glucocorticoid dose from day 10 to day 17after treatment. Approximately one-third of patients no longer requiretransfusion support after treatment with ATG alone.159–161

Of 358 patients responding to immunosuppressive therapy, princi-pally ATG alone, 74 (21%) relapsed after a mean of 2.1 years. The actu-arial incidence of relapse was 35 percent at 10 years.162 Similar resultswere observed when 227 patients were treated with immunosuppres-sion, primarily ATG alone.163 The actuarial survival at 15 years was 38percent following immunosuppression.162 However, a combination ofimmunosuppressive agents provides more effective therapy than ATGalone (see “Combination Immunotherapy” below).

Twenty-eight (22%) of 129 patients treated with ALG developedmyelodysplasia, leukemia, paroxysmal nocturnal hemoglobinuria, orcombined disorders.164 This tendency to relapse and to develop clonalhematologic disorders was reviewed by the European CooperativeGroup for Bone Marrow Transplantation in 468 patients, most ofwhom received ATG.165 The risk of a hematologic complicationincreased continuously and reached 57 percent at 8 years afterimmunosuppressive therapy. A further survey found 42 (5%) malig-nancies in 860 patients treated with immunosuppression, whereas only9 (1%) malignancies were seen in 748 patients who received marrowtransplants.166

Cyclosporine Administration of cyclosporine, a cyclic polypeptide thatinhibits IL-2 production by T lymphocytes and prevents expansion ofcytotoxic T cells in response to IL-2, is another approach to immunother-apy. After the initial report of its ability to induce remission in 1984,167

several groups have used cyclosporine as either (1) primary treat-ment,168–171 (2) in patients refractory to ATG or glucocorticoids,169–174

(3) in combination with granulocyte colony-stimulating factors,175,176 or

(4) in varying combinations with other modes of therapy.177 Cyclospor-ine is administered orally at 10 to 12 mg/kg per day for at least 4 to 6months. Dosage adjustments may be required to maintain troughblood levels of 200 to 400 ng/mL. Renal impairment is common andmay require increased hydration or dose adjustments to keep creatininevalues below 2 mg/dL. Cyclosporine also may cause moderate hyper-tension, a variety of neurological manifestations, and other side effects.Several drug classes interact with cyclosporine to either increase (e.g.,some antibiotics and antifungals) or decrease (e.g., some anticonvul-sants) blood levels. Responses usually are seen by 3 months and mayrange from achieving transfusion independence to complete remission.Approximately 25 percent of patients respond to this agent when usedalone, but the response rate has ranged from 0 to 80 percent in variousreports.177

Although immunosuppression with ALG or ATG has been used thelongest and has a seemingly better response rate, there are certainadvantages to cyclosporine. This drug does not require hospitalizationor use of central venous catheters. Fewer platelet transfusions arerequired during the first few weeks of therapy compared to treatmentwith ALG or ATG. A French cooperative trial showed equal effective-ness of ATG plus prednisone compared to cyclosporine.178 In thiscrossover study of newly diagnosed patients, survival of approximately65 percent was observed 12 months after diagnosis.Combination Immunotherapy Combination treatment of severe aplas-tic anemia usually includes, for example, ATG, 40 mg/kg per day, for 4days; cyclosporine, 10 to 12 mg/kg per day, for 6 months and methyl-prednisolone, 1 mg/kg per day, for 2 weeks.179 The dose of cyclosporineis adjusted to maintain a trough level of 200–400 ng/mL. Prophylaxisfor Pneumocystis carinii with daily trimethoprim-sulfamethoxazole orwith monthly pentamidine inhalations should be considered for thesepatients as they receive immunosuppressive therapy.

The addition of cyclosporine to the combination of ALG and gluco-corticoids improves response rates to approximately 70 percent ofpatients (Table 34–6).180,181 G-CSF added to the combined immuno-suppressive therapy does not increase response rate or survival.183

Response is usually defined as a significant improvement in red cells,white cells, and platelets to eliminate risk of infection and bleeding andthe requirement for red cell transfusions.

TABLE 34–6. Response to Immunotherapy in Patients with Severe Aplastic Anemia

Year of Publication Principal Drugs Used

No. Pts (Age-range, yrs)

Significant Response No. (%)

Survival at 5/10 Years (%)

Relapse at 5 Years (Cum%) Comments Reference

2008 ATG + CYA 77 (<18) 57 (74) 83/80 25 8.5% evolved to clonal myeloid disease 1802007 ATG + CYA 44 (NR) 31 (70) NR/88 NR All cases were associated with hepatitis 1812007 ATG + CYA 47 (19–75) 31 (66) 80/NR 45 No late clonal diseases at 5 years 1822007 ATG + CYA + G-CSF 48 (19–74) 37 (77) 90/NR 15 No late clonal diseases at 5 years 1822006 ATG + CYA 47 (8–71) 37 (79) 80/75 NR No late clonal diseases at 10 years 1832006 ATG + CYA + G-CSF

+ rhuEPO30 (5–68) 22 (73) 80/75 NR One patient developed clonal myeloid

disease 183

ATG, antithymocyte globulin; Cum%, cumulative percent; CYA, cyclosporine A, G-CSF, granulocyte colony-stimulating factor; No. Pts, number of patients; NR, not reported; rhuEPO, recombi-nant human erythropoietin.

NOTE: In some cases response, survival, and relapse percentages are very close approximations, read off the published graphs. Significant response combines complete and partial remissions,which usually means the platelet and red cell count are high enough to avoid transfusions and neutrophil count over a critical level. Some protocols used short periods of glucocorticoid treat-ment to ameliorate reactions to ATG. There is a significant frequency of relapses or progression to clonal myeloid disease after 5 to 10 years postimmunotherapy.


The 5-year survival after completion of combination immunosup-pressive therapy may approximate that after stem cell transplanta-tion.184 Forty-eight children treated between 1983 and 1992 had a 10-year survival of approximately 75 percent for marrow transplantationand approximately 75 percent for combined immunosuppressive ther-apy, although there were only half the number of severely affectedpatients in the immunosuppressive therapy group.185 Thus, immuno-suppression may be preferable for patients who are older than 30 yearsof age and in those who may experience a delay in finding a suitabledonor. Marrow transplants are, however, curative for aplastic anemia,whereas more frequent sequelae have been found after immunosup-pressive therapy,186–188 notably a substantial rate of evolution to amyelodysplastic syndrome or acute myelogenous leukemia.

A recent National Institutes of Health protocol was designed toincrease immune tolerance by specific deletion of activated T lympho-cytes that target primitive hematopoietic progenitor cells.24 Concurrentadministration of cyclosporine with ATG may diminish the ATG effectso that in this program cyclosporine is introduced at a later time. Theaddition of new immunosuppressive agents, such as mycophenolatemofetil, rapamycin, or monoclonal antibodies, to the IL-2 receptor maybe more effective in decreasing cytotoxic T cells, sparing the targetedhematopoietic stem cells.24

For the 30 to 40 percent of patients who relapse after immunother-apy, retreatment with ATG and cyclosporine is effective in 50 to 60 per-cent of them.189,190

High-Dose Glucocorticoid Treatment Marrow recovery can occur aftervery high doses of glucocorticoids.191,192 Methylprednisolone in therange of 500 to 1000 mg daily for 3 to 14 days has been successful, butthe side effects, which include marked hyperglycemia and glycosuria,electrolyte disturbances, gastric irritation, psychosis, increased infec-tions, and aseptic necrosis of the hips, can be severe. Glucocorticoids atlower doses commonly are used only as a component of combinationtherapy for aplastic anemia to ameliorate the toxic effects of ATG andin providing additional lymphocyte suppression.

High-Dose Cyclophosphamide Therapy High-dose cyclophosphamide hasbeen used as a form of immunosuppression.193 Although it would seeminappropriate to administer high doses of chemotherapy to patientswith severe marrow aplasia, this approach was based on observations ofautologous recovery after preparative therapy for allogeneic trans-plants.6 Ten patients received cyclophosphamide at 45 mg/kg per dayintravenously for 4 days with or without cyclosporine for an additional100 days. Gradual neutrophil and platelet recovery ensued over 3months. Seven patients responded completely and remained in remis-sion 11 years after treatment. High-dose cyclophosphamide treatmentmay spare hematopoietic stem cells, which have high levels of aldehydedehydrogenase and are relatively resistant to cyclophosphamide.194,195

Thus, cyclophosphamide in this situation may be more immunosup-pressive than myelotoxic. The most extensive trial of high-dose cyclo-phosphamide resulted in 65 percent of patients responding completelyat 50 months.196 However, the role of this regimen as initial therapy isnot clear because of early toxicity that may exceed that of the ATG-cyclosporine combination.197 The probability of a durable remissionmay be superior, but there are insufficient data (comparative clinicaltrials) to conclude whether high-dose cyclophosphamide provides bet-ter long-term results than ATG and cyclosporine. The latter approach isfavored at this time.

Rituximab A case report of the successful use of the anti-CD20 human-ized mouse antibody rituximab has provided preliminary evidence forits potential effectiveness in treating aplastic anemia.198 Clinical trialsshould examine its efficacy compared to standard immunotherapy(ATG and cyclosporine), in patients refractory to standard therapy, or

as a third drug in an immunotherapy regimen. The role of B lympho-cytes in the pathogenesis of aplastic anemia has not been defined.Androgens Randomized trials have not shown efficacy when andro-gens were used as primary therapy for severe or moderately severeaplastic anemia.199,200

Androgens stimulate the production of erythropoietin, and theirmetabolites stimulate erythropoiesis when added to marrow cultures invitro. High doses of androgens were beneficial in some patients withmoderately severe aplasia.199 Series of patients were reported in whichsurvival seemed improved as compared with historical controls, butthis could have resulted from improved supportive care.136 Masculin-ization and other androgen side effects can be severe. Long-term survi-vors after androgen therapy have essentially the same progression toclonal hematologic disorders as patients treated with immunosuppres-sive agents.136 These agents have been replaced by immunosuppressionor allogeneic hematopoietic stem cell transplantation.Cytokines Despite their effectiveness in accelerating recovery from che-motherapy, these agents have been far less effective in achieving long-term benefits in patients with severe aplastic anemia. Daily treatmentwith G-CSF201,202 has improved marrow cellularity and increased neutro-phil counts approximately 1.5- to 10-fold. Unfortunately, in nearly allpatients, the blood counts return to baseline within several days of cessa-tion of therapy. Although occasional patients show evidence of trilineagemarrow recovery with long-term therapy, the vast majority do notrespond. Therapy with myeloid growth factors is probably best reservedfor episodes of severe infection or as a preventive measure prior to dentalwork or other procedures that would compromise mucosal barriers inpatients who have not responded to stem cell transplant or immunother-apy. Prophylactic use of growth factors is not warranted. G-CSF in a doseof 5 μg/kg by subcutaneous injection is easiest to administer and seemsto be associated with the fewest side effects. The drug can be given dailyor fewer times per week depending on the response. Newer pegylatedpreparations have greater longevity and usually are administered at lessfrequent, every-other-week intervals.

IL-1, a potent stimulator of marrow stromal cell production of othercytokines, and IL-3 have been ineffective in small numbers of patientswith severe aplastic anemia.204,205 These disappointing results withcytokines are not unexpected, as previous work has found high serumlevels of growth factors in patients with aplastic anemia. Moreover, themajority of patients have suppression of very primitive progenitors,which may be unresponsive to individual factors that act on moremature progenitor cells.Splenectomy Removal of the spleen does not increase hematopoiesis butmay increase neutrophil and platelet counts two- to threefold andimprove survival of transfused red cells or platelets in highly sensitizedindividuals.206 The surgical morbidity and mortality in patients with fewplatelets and white cells makes this a questionable therapeutic procedure.Because there are more successful methods of therapy that attack the fun-damental problem, this approach would not be used today.Other Therapy High doses of intravenous gamma globulin have beengiven to small numbers of patients with severe aplastic anemia207,208

because of its success in treating certain cases of antibody-mediatedpure red cell aplasia. Some improvement was noted in 4 of 6 patientstreated. Another treatment that is occasionally successful is lympho-cytapheresis to deplete T cells.209,210

Course and PrognosisAt diagnosis, the prognosis is largely related to the absolute neutrophiland platelet count. The absolute neutrophil count is the most importantprognostic feature, with a count of fewer than 500/μL (0.5 × 109/L) con-sidered severe aplastic anemia and a count of fewer than 200/μL (0.2 ×


109/L) very severe aplastic anemia, the latter associated with a poorresponse to immunotherapy and usually a dire prognosis if early suc-cessful allogeneic transplant is not available. In the past, the prognosisappeared worse when the disease followed hepatitis.69,70 But more com-prehensive results with immunosuppression191 or hematopoietic stemcell transplantation211 show an equivalent response to that seen withidiopathic or drug-induced cases.

Before marrow transplantation and immunosuppressive therapy, morethan 25 percent of the patients with severe aplastic anemia died within 4months of diagnosis; half succumbed within 1 year.212,213 Marrow trans-plantation is curative for approximately 80 to 90 percent of patientsyounger than 20 years of age, approximately 70 percent if between theages of 20 and 40 years, and approximately 50 percent if older than age 40years.151,214 Unfortunately, as many as 40 percent of transplant survivorssuffer the deleterious consequences of chronic graft-versus-host dis-ease,151 and the risk of subsequent cancer can be as high as 10 percent inolder patients or after immunotherapy prior to hematopoietic stem celltransplantation.215 The best outcomes occur in those patients who havean allele-based HLA-matched sibling, have not been exposed to immuno-suppressive therapy prior to transplantation, have not been exposed andsensitized to blood cell products, have had marrow rather than a bloodstem cell donor product, and have not been subjected to high-dose radia-tion in the conditioning regimen for transplantation.151,215–217

Combination immunosuppressive therapy with ATG and cyclosporineleads to a marked improvement in approximately 70 percent of thepatients. Although some patients have normal blood counts, many con-tinue with moderate anemia or thrombocytopenia. In as many as 40 per-cent of patients initially responding to immunosuppressive therapy, theirdisease may relapse or progress over 10 years to paroxysmal nocturnalhemoglobinuria, a myelodysplastic syndrome, or acute myelogenous leu-kemia.162–169,195–197 Moreover, the beneficial effects of immunotherapyare often lost 10 years after treatment. In 168 transplanted patients theactuarial survival at 15 years was 69 percent, and in 227 patients receivingimmunosuppressive therapy it was 38 percent.162

Treatment with high-dose cyclophosphamide produces early resultssimilar to that seen with the combination of ATG and cyclosporine.218

However, cyclophosphamide has greater early toxicity and slowerhematologic recovery, but may generate more durable remissions. Itsuse has been too limited to reach a firm conclusion on its relative mer-its and it is rarely used as first choice immunotherapy.

HEREDITARY APLASTIC ANEMIA

■ FANCONI ANEMIA

Definition and HistoryFanconi anemia is the most common form of constitutional aplasticanemia and was initially described in three brothers by Fanconi in1927.219 It is inherited as an autosomal recessive condition that resultsfrom defects in genes that modulate the stability of DNA.

EpidemiologyFanconi Anemia is an uncommon disorder and is estimated to bepresent in 1 in 1 million individuals. It is far more frequent in Afrikanersof European descent.220 This unusually high frequency has been attrib-uted to a founder effect.

Etiology and PathogenesisThirteen complementation groups, defined by somatic cell hybridiza-tion, are associated with the development of Fanconi anemia.221 A

complementation group is a genetic subgroup. Identifying a comple-mentation group requires adding a gene to the genome of a cell to cor-rect (complement) the genetic defect. This procedure can be done bycell fusion studies. After fusing two cells together, thereby joining theirgenetic material, one can test the cells for the genetic defect. In the caseof Fanconi anemia, this would be with the diepoxybutane test. Hybridsin which the hypersensitivity to diepoxybutane is corrected (comple-mented) can be assumed to result from the fusion of cells from differ-ent genetic subgroups (complementation groups), whereas hybrids thatstill show the sensitivity are the result of fusion of cells from the samesubgroup. Because one can determine the complementation groupwithout knowing the gene involved, this approach is the first step inunderstanding the genetic basis of a disease. Once the genes are known,one does not need to use cell fusion studies; rather, retroviral vectorscan be used to insert corrected genes into the cells.

The complementation groups have been designated FANCA, B, C,D1, D2, E, F, G, I, J, L, M, and N. Table 34–7 lists the gene mutations cor-responding to these complementation groups. The great majority ofpatients have mutations of FANCA, FANCC, or FANCG.222 It has beenproposed that the A and C gene products, which are cytoplasmic pro-teins, form an “FA core complex” with the products of genes B, E, F, G,L, and M, which are adaptors or phosphorylators.222,223 The complextranslocates to the nucleus, where it is required for the ubiquitylation ofFANCD2 and protects the cell from DNA cross-linking and participatesin DNA repair (Fig. 34–4). DNA damage initiates activation of theFA/BRCA pathway and ubiquitylation of FANCD2, which is targeted tothe altered DNA and facilitates repair by interacting with DNA repairproteins, BRCA1, FANCD1/BRCA2, FANCN/PALB2, and RAD51. Inthe presence of a mutant gene product, normal function is disturbedleading to damaging effects in sensitive tissues, including hematopoieticcells. In addition to the genetic defects leading to DNA instability and aninability to repair DNA, TNF-α and -γ are overexpressed in the marrowof Fanconi anemia patients.224 The excess TNF-α may play a role in thesuppression of erythropoiesis in these patients.

Clinical FeaturesGrowth retardation results in short stature, and skeletal anomalies arecommon. Absent, misshapen, or supernumerary thumbs and dysplasticradii occur in half the patients. Hip and vertebral abnormalities alsomay occur. Septal heart defects, eye abnormalities, and absent, mis-shapen, or fused kidneys may be present. Learning disability is fre-quent, and microcephaly and mental retardation may be a feature.Hypogonadism also may be evident. The skin may be generally hyper-pigmented or may have areas of abnormal skin pigmentation referredto as café-au-lait spots, which are flat, light brown, and from 1 to 12centimeters in diameter. Hepatosplenomegaly is not a feature of thedisease. Some patients have no or minor phenotypic abnormalities andmay be diagnosed as a result of the onset of marrow failure or a cancerinvolving any of many sites as late as the fifth decade of life.

The onset of marrow failure is gradual and usually is evident duringthe last half of the first decade of life. The manifestations of anemia,including weakness, fatigue, and dyspnea on exertion, and of thrombo-cytopenia with epistaxis, purpura, or other unexpected bleeding, arethe principal findings. Hematologic and visceral manifestations arecombined eventually in more than a third of patients, but some mayhave cytopenias and inconspicuous somatic changes, whereas othersmay have somatic anomalies with no or a nominal disorder of bloodcell formation for months or years. Some who carry the gene may bevirtually unaffected.225–227 In a review of the more than 1300 patients inthe literature, 100 patients or fewer than 7 percent without anomalieswere identified by chromosome breakage studies (see “Laboratory Fea-tures” below) because of affected siblings.227 In the past, children in


Fanconi families with an onset of aplastic anemia without congenitalsomatic abnormalities were thought to have a different disorder termedEstren-Dameshek syndrome.228 However, these children, whose lym-

phocytes show sensitivity to diepoxybutane, are considered to haveFanconi anemia without skeletal abnormalities.

Laboratory FeaturesBlood counts and marrow cellularity are often normal until 5to 10 years of age, when pancytopenia develops over anextended interval. Macrocytosis with anisocytosis and poikilo-cytosis may be present before any cytopenia occurs. Thrombo-cytopenia may precede the development of granulocytopeniaand anemia. The marrow becomes hypocellular, and in vitrocolony assays reveal a decrease in CFU-GM and BFU–E.227

Random chromatid breaks are present in myeloid cells,lymphocytes, and chorionic villus biopsy samples. This chro-mosome damage is intensified after exposure to DNA cross-linking agents such as mitomycin C or diepoxybutane. Thehypersensitivity of the chromosomes of marrow cells or lym-phocytes to the latter agent is used as a diagnostic test for thiscondition. Cell-cycle progression is prolonged at the G2 to Mtransition, and the cells are more susceptible to oxygen toxicitywhen cultured in vitro. It is important to test the lymphocytesfrom pediatric patients with aplastic anemia for sensitivity todiepoxybutane, because therapy for Fanconi anemia differsfrom that used for acquired aplastic anemia.

In the near future, clinical laboratories will be able to geno-type suspected patients. Determining the specific gene muta-tion responsible in a patient (see Table 34–7) is importantbecause it confirms the diagnosis, identifies the genotypelinked to BRCA2 that may predispose to a cancer (e.g. breast,ovary), and permits carrier detection.229

Differential DiagnosisThe differential diagnosis of Fanconi anemia includes othercauses of aplastic anemia, particularly those familial syndromes

TABLE 34–7. Gene Mutations Found in Fanconi Anemia

Gene Chromosome Location % of Patients Inheritance Protein Function

FANCA 16q24.3 ~65* AR FA Core Complex

FANCB Xp22.31 rare XLR FA Core Complex

FANCC 9q22.3 ~10 AR FA Core Complex

FANCDI (BRCA2) 13q12.3 rare AR RAD51 Recruitment

FANCD2 3p25.3 rare AR Monoubiquitinated protein

FANCE 6p21.3 ~10 AR FA Core Complex

FANCF 11p15 rare AR FA Core Complex

FANCG (XRCC9) 9p13 ~10 AR FA Core Complex

FANCI (KIAA1794) 15q25–26 rare AR Monoubiquitination of FANCD2

FANCJ (BACH1/BRIP1) 17q22.3 rare AR 5′ to 3′ DNA helicase/ATPase

FANCL (PHF9/ POG) 2q16.1 rare AR FA Core Complex, E3 Ubiquitin ligase

FANCM (Hef) 14q21.3 rare AR FA Core Complex, ATPase/translocase, DNA helicase motifs

FANCN (PALB2) 16q12.1 rare AR Regulation of BRCA2 localization

To be identified ~5

AR, autosomal recessive; ATPase, adenosine triphosphatase; XLR, X-linked recessive.

*There are more than 100 mutant FANCA alleles, approximately 40% of which are large intragenic deletions. This table was made using material from references 221–223.

FIGURE 34–4. Representation of the “FA/BRCA pathway.” Following DNA damage when a replica-tion fork encounters a DNA cross-link, ATR (ataxia telangiectasia and rad3-related protein) is activated.This leads to the activation of the FA pathway as well as cell-cycle checkpoint activation via the ATM(Ataxia Telangiectasia Mutated) protein. Activation of the FA pathway leads to the formation of the “FAcore complex” (consisting of the FA proteins A, B, C, E, F, G, L, and M). This activated FA core complexleads to the monoubiquitination of FANCD2 (FANCD2-Ub) and FANCI (I-Ub). The I-Ub/FANCD2-Ubcomplex is then targeted to the chromatin containing the cross link where it interacts with BRCA2 andpossibly other DNA repair proteins (e.g., RAD51, J, N) leading to the repair of the DNA damage. Pro-teins mutated in the different FA subtypes are shown in yellow. (Reproduced from reference 221 withpermission of Elsevier.)

DNAdamage

Stalledreplicationfork

activation

Ub UbATR

ATM

Checkpointresponse

DNA repair

Genomic stability

BRCAI

BRIPI (J)PALB2 (N)

I

I

I

IAGBLCFME

D2

D2 D2

RAD51

BRCA2(D1)

D2

P P

UbUb


associated with skeletal anomalies and other dysmorphic features.Other familial types of aplastic anemia have been reported with orwithout associated anomalies. In those instances in which no sensitivityto DNA damaging agents is observed, the syndrome does not representFanconi anemia. Several uncommon syndromes of this type aredescribed below and are tabulated in Table 34–8.

Therapy and CourseMost patients with Fanconi anemia do not respond to ATG or cyclo-sporine but do improve with androgen preparations, often for as longas several years. Cytokines may provide some improvement in bloodcounts, but their effect may wane. Studies in a mouse model also sug-gest that cytokine effects may not be sustained.230 The cumulativemedian survival is about 20 years from progressive marrow failure,conversion to myelodysplastic syndrome, acute myelogenous leukemia(approximately 10 percent of patients), or the development of a varietyof other cancers such as genitourinary system, digestive system (espe-cially liver), head and neck.231 Multiple cancers in an individual patientalso occur. Cancers may occur as late as the fifth decade of life and pre-cede the diagnosis of Fanconi anemia in 25 percent of patients.231 Thepresence of a clonal cytogenetic abnormality or marrow morphologyconsistent with myelodysplasia markedly reduces the 5-year survival.232

Allogeneic hematopoietic stem cell transplantation is curative for themarrow manifestations of Fanconi anemia.232–235 A marked reductionin dosage of the marrow-conditioning regimen of cyclophosphamideand radiation is necessary owing to the undue sensitivity of the tissuesto DNA-damaging exposures. The risk of cancer is so high that, wherepractical, surveillance should be used, for example, frequent pelvic

exams in females, hepatic ultrasonography to detect adenomas, andcareful oropharyngeal examinations. Therapy of cancer in patients withFanconi anemia needs to consider the marked sensitivity of their cellsto DNA cross-linking agents and radiotherapy.

Normal cDNA has been transferred into cells from patients with res-toration of resistance to DNA damaging agents.236,237 Difficulties in thisapproach include the paucity of stem cells in these patients, as well asthe potential toxicity of the gene transfer methodology.

■ DYSKERATOSIS CONGENITA

DefinitionThis inherited disorder is characterized by cutaneous and mucous mem-brane abnormalities, progressive marrow insufficiency, and a predisposi-tion to malignant transformation. It is much more common in malesthan in females, and occurs in about 1 per 1 million population.221,238

PathogenesisDyskeratosis usually is inherited as a recessive X-chromosome–linkeddisorder although rare cases can have autosomal dominant or autoso-mal recessive inheritance (Table 34–9). The disease is a reflection oftelomere complex dysfunction,239,240 and it results from defective telomer-ase activity resulting from mutations in the telomerase-related genes (Fig.34–5).240 The telomerase complex maintains the length of telomeres,which are nucleotide tandem repeat structures residing at the termini ofeukaryotic chromosomes (e.g., 5′-TTAGGG-3′). Telomerase restores theG-rich telomere repeats that are lost as a result of end-processing during

TABLE 34–8. Other Rare Syndromes Associated with Aplastic Anemia

Disorder Findings Inheritance Mutated Gene References

Ataxia-pancytopenia (myelocerebellar disorder)

Cerebellar atrophy and ataxia; aplastic pancytopenia; ± monosomy 7; increased risk of AML

AD Unknown 256–258

Congenital amegakaryocytic thrombocytopenia

Thrombocytopenia; absent or markedly decreased marrow megakaryocytes; hemorrhagic propensity; elevated thrombopoietin; propensity to progress to aplastic pancytopenia; propensity to evolve to clonal myeloid disease

AR (compound heterozygotes)

MPL 259, 260

DNA ligase IV deficiency Pre- and postnatal growth delay; dysmorphic facies; aplastic pancytopenia

AR (compound heterozygotes)

LIG4 261–263

Dubowitz syndrome Intrauterine and post-partum growth failure; short stature; microcephaly; mental retardation; distinct dysmorphic facies; aplastic pancytopenia; increased risk of AML and ALL

AR Unknown 264, 265

Nijmegen breakage syndrome Microcephaly; dystrophic facies; short stature; immunodeficiency; radiation sensitivity; aplastic pancytopenia; predisposition to lymphoid malignancy

AR NBS1 266, 267

Reticular dysgenesis (type of severe immunodeficiency syndrome)

Lymphopenia; anemia and neutropenia; corrected by hematopoietic stem cell transplantation

XLR Unknown 268, 269

Seckel syndrome Intrauterine and post- partum growth failure; microcephaly; characteristic dysmorphic facies (bird-headed profile); aplastic pancytopenia; ? increased risk of AML

AR ATR (and RAD3-related gene); PCNT

270–273

WT syndrome Radial/ulnar abnormalities; aplastic pancytopenia; increased risk of AML AD Unknown 274

AD, autosomal dominant; ALL, acute lymphocytic leukemia; AML, acute myelogenous leukemia; AR, autosomal recessive; XLR, X-linked recessive.

NOTE: The listed clinical findings in each syndrome are not comprehensive. The designated clinical findings may not be present in all cases of the syndrome. Isolated cases of familial aplasticanemia with or without associated anomalies that are not consistent with Fanconi anemia or other defined syndromes have been reported.227


normal cell division. Combined with protein, located at the ends ofchromosomes, they maintain chromosome integrity by preventing end-to-end chromosome fusion, preventing chromosome degradation, andpreventing chromosome instability.

In dyskeratosis congenita, the telomeres are markedly shortenedresulting in genomic instability and cell (including marrow cell) apopto-sis. Rapidly proliferating cells are at highest risk for dysfunction. Muta-tions of the DKC1 gene are responsible for the X-linked recessive form.DKC1 encodes dyskerin, which is a conserved multifunctional proteincomponent of the telomerase complex. Mutations of the TERT, TERC,and TINF2 genes are the principal abnormalities in the autosomal domi-nant form. TERC is the RNA component of the telomerase reverse tran-scriptase that TERT, the reverse transcriptase, uses to synthesize the 6-bprepeats on the 3′ end of telomeric DNA. TINF2 is a component of theshelterin complex. The latter permits the distinction of telomeres from

sites of DNA damage, preventing their otherwise inappropriate process-ing. Recessive mutations in NOP10, which encodes small ribonucleopro-teins associated with the telomerase complex, have been described in aconsanguineous family.241 Homozygous recessive mutations in the telo-merase reverse transcriptase (TERT) produce a severe variant, referred toas the Høyeraal-Hreidarsson syndrome.275

Clinical FindingsThe cutaneous findings usually appear after 5 years of age and includereticulated, tan to gray, hyperpigmented and hypopigmented cutaneousmacules; alopecia of scalp, eyelashes, and eyebrows; adermatoglyphia(loss of dermal ridges on fingers and toes); hyperkeratosis of palms andsoles; mucosal leukoplakia in 75 percent of patients; and dystrophicnails in more than 85 percent of patients.221,238,239 Other mucosal sites,such as conjunctiva, lacrimal duct, esophagus, urethra, vagina, and

anus, can be involved, sometimes with stenosis and,for example, dysphagia or dysuria. Pulmonary vascu-lar involvement occurs in a significant minority ofaffected children. Aplastic anemia usually develops inlate childhood or early adulthood and is evident in theclassical blood and marrow findings described underacquired aplastic anemia, above. Female carriers of X-linked dyskeratosis congenital may have slight abnor-malities such as a dystrophic nail, a single area ofhypopigmentation, or slight leukoplakia.238

DiagnosisThe diagnosis results from the combination of pheno-typic findings and blood cell deficiencies. Geneticanalysis for telomerase complex gene mutationsshould be used to confirm the clinical conclusion.Shortened telomere length in leukocytes also can beassessed by flow cytometric fluorescence in situhybridization studies.242

ManagementStem cell hematopoietic transplantation has hadinconsistent results because of frequent and severeposttransplantation complications.243 Nonmyeloabla-tive transplantation might improve results.244,245 Trans-plantation might improve the cytopenias but not theabnormalities of other organs or the frequency of sec-ondary nonhematopoietic cancer.

TABLE 34–9. Gene Mutations in Dyskeratosis Congenita

Gene Chromosome Location % of Patients Inheritance Protein Function

DKC1 Xq28 30 XLR Essential part of snoRNPs and telomerase

TERC 3q26 <5 AD RNA 3′ end processing and stability

TERT 5p15.33 <6 AD, AR Reverse transcriptase component of telomerase

NOP10 (NOLA3) 15q14-q15 <1 AR RNA binding

TINF2 14q11.2 11 AD ? Binds to TRF1 to regulate telomere length

To be identified ~50%

AD, autosomal dominant; AR, autosomal recessive; XLR, X-linked recessive.

NOTE: Table prepared from data in references 221, 223, and 273. Percent of patients is approximate because of continuing identification of mutations.

FIGURE 34–5. Representation of the interaction between dyskerin and the other molecules (GAR1, NHP2,NOP10, TERC, and TERT) of the telomerase complex (and their association with different disease categories).Telomerase is an RNA-protein complex because TERC is an RNA molecule that is never translated. The othermolecules (dyskerin, GAR1, NHP2, NOP10 and TERT) are proteins. The minimal active telomerase enzyme iscomposed of two molecules each of TERT, TERC, and dyskerin. Dyskerin, GAR1, NHP2, and NOP10 are impor-tant for the stability of the telomerase complex. AA, aplastic anemia; AD–DC, autosomal dominant dyskerato-sis congenita; AR–DC, autosomal recessive dyskeratosis congenita; AR–HH, autosomal recessive Høyeraal-Hreidarsson syndrome; MDS, myelodysplasia; PNH, paroxysmal nocturnal hemoglobinuria; PF, pulmonaryfibrosis; X-linked DC, X-linked dyskeratosis congenita; X-linked HH, X-linked Høyeraal-Hreidarsson syndrome.(Reprinted from reference 221 with permission of Elsevier.)

TERT: heterozygous mutations in AA, a disease resembling AD–DC and PF

Homozygous mutations in classical AR–DC and AR–HH

NHP2

GAR1

NOP10: homozygous mutation in AR–DC

The dyskerin complex: also involved in processing ribosomal and small nuclear RNAs

TERC: heterozygousmutations in AD–DC, AA,MDS, PNH, and PF

Dyskerin: hemizygousmutations in X-linked DCand X-linked HH


Course and PrognosisThe incidence of squamous cell carcinoma of mucosal sites is increasedand they often originate in sites of leukoplakia in the skin, gastrointesti-nal, or genitourinary tracts. These usually develop between the ages of20 and 30 years. Mortality from neutropenic infection or thrombocyto-penic hemorrhage occurs in about two-thirds of patients with aplasticanemia. Median survival is about 30 years.

■ SHWACHMAN-DIAMOND SYNDROMEDefinitionAn uncommon inherited disorder that is estimated to occur once inevery 100,000 births, manifesting exocrine pancreatic insufficiencywith secondary steatorrhea, blood cell deficiencies, and skeletal abnor-malities. It was first described in 1964.247,249

PathogenesisShwachman-Diamond syndrome results from mutations in the SBDSgene on chromosome 7q11, which induces accelerated cellular apoptosisvia the FAS pathway.249 The resulting hyperproliferation may account forthe abnormal telomere shortening that has been documented in the leu-kocytes in this condition.250 The pathogenetic mechanism that (1) pre-vents development of pancreatic acinar cells, (2) results in abnormal bonemorphogenesis, and (3) causes marrow impairment of blood cell produc-tion is not understood. SBDS knockdown in experimental animals affectsexpression of genes involved in brain, bone, and marrow development,and may be the result of the gene’s role in RNA processing.251,252 Themutations also result in abnormalities in neutrophil motility and chemo-taxis, but pus formation in vivo seems adequate.

Clinical FindingsPancreatic insufficiency, steatorrhea, and neutropenia are present inmost patients at the time of diagnosis.247,248 Pallor may reflect anemiaand easy bruising; epistaxis or bleeding from other sites reflect throm-bocytopenia. Neutropenia occurs in approximately 95 percent, ane-mia in approximately 50 percent, and thrombocytopenia inapproximately 35 percent of patients.249 Thus, a substantial pluralityof patients has bicytopenia or tricytopenia with an hypoplastic mar-row. Fetal hemoglobin levels are elevated in approximately 75 percentof the patients, perhaps secondary to erythroid hypoplasia. Cytoge-netic abnormalities involving chromosomes 7 and 20 have beendescribed in marrow cells. Nutritional inadequacies related to intesti-nal malabsorption result in a failure to thrive. Short stature is charac-teristic. Skeletal abnormalities are present in most patients, notablyosteopenia, but also syndactyly, supernumerary metatarsals, coax veradeformity, and dental enamel defects and caries. Delayed puberty iscommon. The neutropenia and chemotactic abnormality may resultin recurrent infections, including sinusitis, otitis, pneumonia, osteo-myelitis, and others. Pancreatic cell lipase production improves withage, and as many as half the patients may have improvement in lipidabsorption in the small bowel with time.

ManagementSupportive care, particularly with supplemental pancreatic enzymes, toprovide proper nutrition, and appropriate and prompt treatment ofbacterial infections with antibiotics is important. Many agents, includ-ing G-CSF, glucocorticoids, pancreatic extract, vitamins, have beentried to improve the neutropenia with erratic results. Some agents havepotential risks, such as G-CSF fostering clonal evolution and glucocor-ticoids fostering immunodeficiency. Severe hematopoietic dysfunction

and cytopenias can be corrected with allogeneic hematopoietic stemcell transplantation.253

Course and PrognosisDeath from overwhelming sepsis is common. These patients, especiallymales, have a significant risk of progression to a myelodysplastic syn-drome or acute myelogenous leukemia.248,254,255 Survival is a functionof the severity of the cytopenias. If the cytopenias are mild, survival isnot uncommon into the fourth or fifth decade of life. If symptomaticpancytopenia, especially neutropenia, is present, median survival isabout 20 to 30 years.248,255

■ OTHER INHERITED APLASTIC ANEMIASSeveral other rare syndromes are associated with aplastic pancytopenia,and these are described in Table 34–8. Congenital amegakaryocyticthrombocytopenia results from mutations in the thrombopoietinreceptor gene, MPL.259,260 Reticular dysgenesis results from a pluripo-tential stem cell defect as both lymphoid and myeloid progenitors areaffected.268,269 The Seckel syndrome results from mutations in the ATRgene, and marrow cells exhibit heightened sister chromatid exchange.270–273

The ataxia-telangiectasia mutated and rad3-related (ATR) kinaseorchestrates cellular responses to DNA damage and replication stress.The genetic basis of marrow failure in these four syndromes thatinvolve aplastic pancytopenia is not yet known, but may be related todefects in the ATR-dependent DNA damage-repair pathway.259 Most ofthese syndromes can be treated by marrow transplantation, but thisstep, if successful, does not correct somatic abnormalities, only thehematopoietic defect. The restoration of robust hematopoiesis by trans-plantation may decrease their propensity to undergo clonal evolution toa clonal myeloid or, in some cases, lymphoid disorder.

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