Chapter 19 - Bone Marrow Failure

PATHOPHYSIOLOGY OF BONE

MARROW FAILURE

Bone marrow failure is the reduction or cessation of blood cell production affecting one or more cell lines. Pancytopenia, or decreased numbers of circulating red blood cells (RBCs), white blood cells (WBCs), and platelets, is seen in most cases of bone marrow failure, particularly in severe or advanced stages.

The pathophysiology of bone marrow failure includes

(1) destruction of hematopoietic stem cells as a result of injury

by drugs, chemicals, radiation, viruses, or autoimmune mecha-

nisms; (2) premature senescence and apoptosis of hematopoi-

etic stem cells as a result of genetic mutations; (3) ineffective

hematopoiesis caused by stem cell mutations or vitamin B12 or

folate deficiency; (4) disruption of the bone marrow microen-

vironment that supports hematopoiesis; (5) decreased produc-

tion of hematopoietic growth factors or related hormones; and

(6) loss of normal hematopoietic tissue as a result of infiltration

of the marrow space with abnormal cells.

Clinical consequences of bone marrow failure vary, depend-

ing on the extent and duration of the cytopenias. Severe pancy-

topenia can be rapidly fatal if untreated. Some patients may

initially be asymptomatic, and their cytopenia may be detected

during a routine blood examination. Thrombocytopenia can

result in bleeding and increased bruising. Decreased RBCs and

hemoglobin can result in fatigue, pallor, and cardiovascular

complications. Sustained neutropenia increases the risk of life-

threatening bacterial or fungal infections.

This chapter focuses on aplastic anemia, a bone marrow

failure syndrome resulting from damaged or defective stem

cells (mechanisms 1 and 2 listed earlier). Bone marrow failure

resulting from other mechanisms may present similarly to

aplastic anemia, and differentiation is discussed later. Because

there are many mechanisms involved in the various bone mar-

row failure syndromes, accurate diagnosis is essential to ensure

appropriate treatment.

APLASTIC ANEMIA

Aplastic anemia is a rare but potentially fatal bone marrow

failure syndrome. In 1888 Ehrlich provided the first case report

of aplastic anemia involving a patient with severe anemia, neu-

tropenia, and a hypocellular marrow on postmortem examina-

tion.1 The name aplastic anemia was given to the disease by

Vaquez and Aubertin in 1904.2 The characteristic features of

aplastic anemia include pancytopenia, reticulocytopenia, bone

marrow hypocellularity, and depletion of hematopoietic stem

cells (Box 19.1). Approximately 80% to 85% of aplastic anemia cases are acquired, whereas 15% to 20% are inherited/congenital.3

Box 19.2 provides an etiologic classification.3-5

Acquired Aplastic Anemia

Acquired aplastic anemia is classified into two major categories:

idiopathic and secondary. Idiopathic acquired aplastic anemia

has no known cause. Secondary acquired aplastic anemia

is associated with an identified cause. Approximately 70%

of all aplastic anemia cases are idiopathic, whereas 10% to 15%

are secondary.3 Idiopathic and secondary acquired aplastic

anemia have similar clinical and laboratory findings. Pa-

tients may initially present with macrocytic or normocytic

anemia and reticulocytopenia. Pancytopenia may develop

slowly or progress at a rapid rate, with complete cessation of

hematopoiesis.

Incidence

In North America and Europe the annual incidence is approxi-

mately 1 in 500,000.6 In Asia and East Asia the incidence is two to three times higher than in North America or Europe, which

may be due to environmental and/or genetic differences.7

Aplastic anemia can occur at any age, with peak incidence at

15 to 25 years and the second highest frequency at older than

60 years.4,6,8 There is no gender predisposition.6

Etiology

As the name indicates, the cause of idiopathic aplastic anemia is

unknown. Secondary aplastic anemia is associated with expo-

sure to certain drugs, chemicals, radiation, or infections. Cyto-

toxic drugs, radiation, and benzenes are responsible for 10% of

secondary aplastic anemia cases and suppress the bone marrow

in a predictable, dose-dependent manner.4,5 Depending on the

dose and exposure duration, the bone marrow generally recov-

ers after withdrawal of the agent. Alternatively, approximately

70% of cases of secondary aplastic anemia occur as a result of

idiosyncratic reactions to drugs or chemicals. In idiosyncratic

reactions the bone marrow failure is unpredictable and unre-

lated to dose.4 Documentation of a responsible factor or agent

in these cases is difficult because evidence is primarily circum-

stantial and symptoms may occur months or years after

exposure. Some drugs associated with idiosyncratic secondary

aplastic anemia are listed in Box 19.3.

4,8

Generally, idiosyncratic secondary aplastic anemia is a rare

event and likely is due to a combination of genetic and environ-

mental factors in susceptible individuals. There are no readily

available tests that predict individual susceptibility to these id-

iosyncratic reactions. However, genetic variations in immune response pathways or metabolic enzymes may play a role.4

There is an approximately twofold higher incidence of human

leukocyte antigen-DR2 (HLA-DR2) and its major serologic

split, HLA-DR15, in aplastic anemia patients compared with

the general population, but the relationship of this finding to

disease pathophysiology has not been elucidated.9,10 There are

also reports that genetic polymorphisms in enzymes that me-

tabolize benzene increase susceptibility to toxicity, even at low

exposure levels.4,11 These include polymorphisms in glutathi-

one S-transferase (GST) enzymes (GSTT1 and GSTM1), my-

eloperoxidase, nicotinamide adenine dinucleotide phosphate

(reduced form, NADPH), quinine oxidoreductase 1, and cyto-

chrome oxidase P450 2E1.11 A deficiency in GST as a result of

the GSTT1 null genotype is overrepresented in Caucasians,

Hispanics, and Asians with aplastic anemia, with a frequency of

30%, 28%, and 75%, respectively.12 Caucasian patients with

aplastic anemia also have a higher frequency (22%) of the

GSTM1/GSTT1 null genotype than the general population.12

GST is important for metabolism and neutralization of chemi-

cal toxins, and deficiencies of this enzyme may increase the risk

of aplastic anemia. Further study is required to assess how these

genetic variations, and other yet undiscovered factors, contrib-

ute to aplastic anemia.

Acquired aplastic anemia occurs occasionally as a complica-

tion of infection with Epstein-Barr virus, human immunodefi-

ciency virus (HIV), hepatitis virus, and human parvovirus

B19.4 In 2% to 10% of patients with acquired aplastic anemia,

there is a history of acute nontypable hepatitis (non-A, non-B,

etc.) occurring 1 to 3 months before the onset of pancytopenia;

this is thought to represent autoimmune hepatitis.13 The ac-

quired aplastic anemia in these cases may be mediated by such

mechanisms as interferon-g (IFN-g) and cytokine release.13

Aplastic anemia associated with pregnancy is a rare occur-

rence, with fewer than 100 cases reported in the literature.14

Approximately 10% of individuals with acquired aplastic ane-

mia have a concomitant autoimmune disease15 and approxi-

mately 10% develop hemolytic or thrombotic manifestations of

paroxysmal nocturnal hemoglobinuria (PNH).16 The overlap

between acquired aplastic anemia and PNH is discussed later.

Pathophysiology

The primary lesion in acquired aplastic anemia is a quantitative

and qualitative deficiency of hematopoietic stem cells. Stem

cells of patients with acquired aplastic anemia have diminished

colony formation in methylcellulose cultures.17 The hemato-

poietic stem and early progenitor cell compartment is identified

by expression of CD34 surface antigens. The CD341 cell popu-

lation in the bone marrow of patients with acquired aplastic

anemia can be 10% or lower than that seen in healthy individu-

als.17 In addition, these CD341 cells have increased expression

of Fas receptors that mediate apoptosis and increased expres-

sion of apoptosis-related genes.18-20

Bone marrow stromal cells are functionally normal in ac-

quired aplastic anemia. They produce normal or even increased

quantities of growth factors and are able to support the growth

of CD341 cells from healthy donors in culture and in vivo after

transplantation.4,21 Individuals with aplastic anemia also have elevated serum levels of erythropoietin, thrombopoietin, gran-

ulocyte colony-stimulating factor (G-CSF), and granulocyte-

macrophage colony-stimulating factor (GM-CSF).22 In addi-

tion, serum levels of FLT3 ligand, a growth factor that stimulates

proliferation of stem and progenitor cells, is up to 200 times

higher in patients with severe aplastic anemia compared with

healthy controls.22,23 However, despite their elevated levels,

growth factors are generally unsuccessful in correcting the cyto-

penias found in acquired aplastic anemia.

The severe depletion of hematopoietic stem and progenitor

cells from the bone marrow may be due to direct damage to

stem cells, immune damage to stem cells, or other unknown

mechanisms. Direct damage to stem and progenitor cells results

from deoxyribonucleic acid (DNA) injury after exposure to

cytotoxic drugs, chemicals, radiation, or viruses.4

Immune damage to stem cells results from exposure to

drugs, chemicals, viruses, or other agents that cause an autoim-

mune cytotoxic T lymphocytic destruction of stem and pro-

genitor cells.24 An autoimmune pathophysiology was first sug-

gested in the 1970s when aplastic anemia patients undergoing

pretransplant immunosuppressive conditioning had an im-

provement in cell counts.25 Further evidence supporting an

autoimmune pathophysiology include (1) elevated blood and

bone marrow cytotoxic (CD81) T lymphocytes with an oligo-

clonal expansion of specific T cell clones26; (2) increased T cell

production of such cytokines as IFN-g and tumor necrosis

factor-a (TNF-a), which inhibit hematopoiesis and induce

apoptosis27-29; (3) upregulation of T-bet, a transcription factor

that binds to the promoter of the IFN-g gene30; (4) increased

TNF-a receptors on CD341 cells31; and (5) improvement in

cytopenias after immunosuppressive therapy (IST).4,24 Approx-

imately two-thirds of patients with acquired aplastic anemia

respond to IST.32 The nonresponders may have a severely de-

pleted stem cell compartment or other pathophysiologic factors

contributing to their cytopenias.32

Possible autoimmune mechanisms include mutation of stem

cell antigens and disruption of immune regulation. Young and

colleagues showed that environmental exposures may alter self-

proteins, induce expression of abnormal or novel antigens,

or induce an immune response that cross-reacts with self-

antigens.24,28 Solomou and colleagues. demonstrated that

CD41CD251FOXP31 regulatory T cells are decreased in aplastic

anemia.33 These regulatory T cells normally suppress autoreac-

tive T cells, and a deficit of these cells may facilitate an auto-

immune reaction. Furthermore, a number of individuals

with aplastic anemia have single nucleotide polymorphisms in

IFN-g/1874 TT, TNF-a/–308 AA, transforming growth factor-

b1/–509 TT, and interleukin-6/–174 GG.34 These polymorphisms

result in cytokine overproduction and may impart a genetic sus-

ceptibility to aplastic anemia as well as contribute to its severity.34

The specific antigens responsible for triggering and sustain-

ing the autoimmune attack on stem cells are unknown. Candi-

date antigens have been identified from aplastic anemia patient

sera, including kinectin,35 diazepam-binding inhibitor-related

protein 1,36 and moesin.37 These proteins are expressed in he-

matopoietic progenitor cells, but their role in the pathogenesis

of aplastic anemia requires further investigation.

Approximately one-third of patients with acquired aplastic

anemia have shortened telomeres in their peripheral blood

granulocytes compared with age-matched controls.38,39 Telomeres

protect the ends of chromosomes from damage and erosion,

and cells with abnormally short telomeres undergo prolifera-

tion arrest and premature apoptosis. Telomerase is an enzyme

complex that repairs and maintains telomeres. Approximately

10% of patients with acquired aplastic anemia and shortened

telomeres have a mutation in the telomerase complex gene for

either the ribonucleic acid (RNA) template (TERC) or the re-

verse transcriptase (TERT).39-41 The cause for shortened telo-

meres in the other 90% of patients may be due to stress hema-

topoiesis or other yet unidentified mutations.39 In stress

hematopoiesis there is an increase in progenitor cell turnover,

and the telomeres become shorter with each cell division.

Approximately 4% of patients with acquired aplastic anemia

and shortened telomeres have mutations in the Shwachman-

Bodian-Diamond syndrome (SBDS) gene.42 The SBDS gene

product is involved in ribosome biogenesis, and its relationship

to telomere maintenance is currently unknown.42 TERT/TERC

and SBDS mutations also occur in the inherited bone marrow

failure syndromes dyskeratosis congenita (DC) and SBDS, re-

spectively, and some patients diagnosed with acquired aplastic

anemia who have these mutations may actually have DC or

SBDS.3,4 Correct differentiation between acquired aplastic

anemia and inherited bone marrow failure syndromes has im-

portant implications for appropriate treatment and prognosis.

Immunosuppressive therapy is not nearly as effective in inher-

ited bone marrow failure compared with acquired aplastic

anemia. Furthermore, hematopoietic stem cell transplantation,

the only known curative treatment for DC and SBDS and a

treatment option for acquired aplastic anemia, should not be

performed with HLA-matched siblings who test positive for

the same genetic mutation.39 Shortened telomeres occur more

often in patients whose pancytopenia does not respond to im-

munosuppressive therapy.43 Defective telomere maintenance

may be another pathophysiologic mechanism of stem cell in-

jury, imparting susceptibility to aplastic anemia after an envi-

ronmental insult.39,41

Clinical Findings

Symptoms vary in acquired aplastic anemia, ranging from as-

ymptomatic to severe. Patients usually present with symptoms

of insidious-onset anemia, with pallor, fatigue, and weakness.

Severe and prolonged anemia can result in serious cardiovascu-

lar complications, including tachycardia, hypotension, cardiac

failure, and death. Symptoms of thrombocytopenia are also

varied and include petechiae, bruising, epistaxis, mucosal bleed-

ing, menorrhagia, retinal hemorrhages, intestinal bleeding, and

intracranial hemorrhage. Fever and bacterial or fungal infec-

tions are unusual at initial presentation but may occur after

prolonged periods of neutropenia. Splenomegaly and hepato-

megaly are typically absent.

Laboratory Findings

Pancytopenia is typical, although initially only one or two

cell lines may be decreased. The absolute neutrophil count is decreased, and the absolute lymphocyte count may be normal

or decreased. The hemoglobin is usually less than 10 g/dL, the

mean cell volume (MCV) is increased or normal, and the per-

cent and absolute reticulocyte counts are decreased. Table 19.1

lists the diagnostic criteria for aplastic anemia by degree of

severity.6,8,44,45

Neutrophils, monocytes, and platelets are decreased in the

peripheral blood, and the RBCs are macrocytic or normocytic

(Figure 19.1). Toxic granulation may be observed in the neutro-

phils, but the RBCs and platelets are usually normal in appear-

ance. Leukemic blasts and other immature blood cells are charac-

teristically absent. The serum iron level and percent transferrin

saturation may be increased, which reflects decreased iron use for

erythropoiesis. Liver function test results may be abnormal in

cases of hepatitis-associated aplastic anemia.

Approximately two-thirds of patients have small numbers

(less than 25%) of PNH clones in the peripheral blood,46 but

only 10% of patients develop a sufficient number of PNH cells

to have the clinical and biochemical manifestations of PNH disease.16 PNH is characterized by an acquired stem cell muta-

tion resulting in lack of the glycosylphosphatidylinositol (GPI)-

linked proteins CD55 and CD59. The absence of CD55 and

CD59 on the surface of the RBCs renders them more suscepti-

ble to complement-mediated cell lysis. It is important to test for

PNH in acquired aplastic anemia because of the increased risk

of hemolytic or thrombotic complications (Chapter 21). His-

torically, PNH diagnosis depended on the Ham acid hemolysis

test: Patients’ cells were placed in acidified serum, and a positive

result demonstrated lysis of RBCs. However, this test was poorly

sensitive, because complement-mediated hemolysis was de-

tected only in the presence of large numbers of circulating

PNH cells. The Ham test has been replaced by flow cytometric

analysis for proteins linked to the GPI anchor; CD55 and CD59

on RBCs, and CD24 and CD14 on granulocytes and mono-

cytes.8,46,47 In addition, flow cytometry for the GPI anchor

(FLAER assay) is the newest and most sensitive assay for detect-

ing PNH cells among granulocytes (Chapter 21).48

Bone marrow aspirate and biopsy specimens have promi-

nent fat cells with areas of patchy marrow cellularity. Biopsy

specimens are required for accurate quantitative assessment of

marrow cellularity, and severe hypocellularity is a characteristic

feature of aplastic anemia (Figure 19.2). Erythroid, granulo-

cytic, and megakaryocytic cells are decreased or absent. Dyserythropoiesis may be present, but there is typically no dys-

plasia of the granulocyte or platelet cell lines. Blasts and other

abnormal cell infiltrates are characteristically absent. Reticulin

staining is usually normal.

In patients receiving immunosuppressive therapy, the risk

of developing an abnormal karyotype is 14% at 5 years and

20% at 10 years.49 Monosomy 7 and trisomy 8 are the most

common cytogenetic abnormalities.47,49 Cytogenetic analysis

using conventional culture techniques often underestimates the

incidence of karyotype abnormalities because of bone marrow

hypocellularity and scarcity of cells in metaphase.50 Alterna-

tively, interphase fluorescence in situ hybridization (FISH) us-

ing DNA probes for specific chromosome abnormalities may be

used. In comparison to conventional cytogenetic analysis, FISH

has greater sensitivity in the detection of chromosome abnor-

malities and can also be performed using nondividing cells.50

In a study performed by Kearns and colleagues, FISH detected

monosomy 7 or trisomy 8 in 26% of aplastic anemia patients who

had a normal karyotype by conventional cytogenetic testing.50

Patients with an inherited bone marrow failure syndrome

may be misdiagnosed with acquired aplastic anemia if symp-

toms manifest in late adolescence or adulthood or if the pa-

tients lack the typical clinical and physical characteristics of an

inherited marrow failure syndrome (e.g., abnormal thumbs,

short stature).3,4 Consideration of inherited bone marrow fail-

ure syndromes in the differential diagnosis of acquired aplastic

anemia is essential because these conditions require a different

therapeutic approach. The inherited bone marrow failure syn-

dromes are discussed later in the chapter.

Treatment and Prognosis

Severe acquired aplastic anemia requires immediate attention

to prevent serious complications. If a causative agent is identi-

fied, its use should be discontinued. Blood product replacement

should be given judiciously to avoid alloimmunization.8 Plate-

lets should not be transfused at counts greater than 10,000/mL,

unless the patient is bleeding.8

One of the most important early decisions is determining

whether the patient is a candidate for bone marrow transplan-

tation (BMT). BMT is the treatment of choice for patients with

severe aplastic anemia who are younger than 40 years of age and

have an HLA-identical sibling.4,8 Unfortunately, only 20% to

30% of patients meet these criteria.4 Therefore, IST, consisting

of antithymocyte globulin and cyclosporine, is used for patients

older than 40 years of age and for patients without an HLA-

identical sibling.8,16 Antithymocyte globulin decreases the num-

ber of activated T cells, and cyclosporine inhibits T cell func-

tion, thereby suppressing the autoimmune reaction against the

stem cells. For patients with severe acquired aplastic anemia

who are not responsive to IST, BMT from a high-resolution

HLA-matched unrelated donor is an option.8,51 With advances

in modern transplantation protocols, overall survival rates are

now only slightly lower with high-resolution HLA-matched

unrelated donors compared with HLA-matched siblings.4 The

response rate for a second course of IST is approximately 65%

for those who experienced relapse and only 30% for those

whose disorder was initially refractive to IST.52 Individuals with

PNH cells (CD55– CD59–) are almost twice as likely to respond

to IST than are those who lack these cells.46 In addition, the

presence of both PNH cells and HLA-DR2 increases the likeli-

hood of response by 3.5-fold.53 G-CSF, other hematopoietic

growth factors, and steroids do not increase overall survival or

improve the response rate; therefore they are not recommended

for routine use.8,54,55

There have been promising results from a phase 1/2 study

evaluating the addition of eltrombopag, a thrombopoietin mi-

metic, to standard IST for severe aplastic anemia, with higher

hematologic response in study patients compared with histori-

cal cohorts.56 Eltrombopag’s mechanism of action in aplastic

anemia is unclear but may be due to stimulatory effects on stem

and progenitor cells. Further studies are currently underway to

evaluate the efficacy of eltrombopag in pediatric severe aplastic

anemia.

Other supportive therapy includes antibiotic and antifungal

prophylaxis in cases of prolonged neutropenia. Patients with

mild to moderate aplastic anemia may not require treatment

but must be monitored periodically for pancytopenia and

abnormal cells.

The overall outcome for patients with acquired aplastic ane-

mia has dramatically improved in the past two decades. Chil-

dren have higher survival rates compared with adults with both

BMT and IST as first-line treatment.51 With BMT, the 10-year

survival for those aged 1 to 20 and those older than 40 years is

86% and 56%, respectively; with IST, 10-year survival for those

same age groups is 84%, and 58%, respectively.51 Additional

outcomes in the IST-treated patients include a 10-year risk of

developing hemolytic or thrombotic PNH and a 10% to 20%

risk of myelodysplastic syndrome (MDS) or leukemia.16,47 De-

velopment of monosomy 7 predicts poor outcome, with a

greater likelihood of unresponsiveness to IST and progression

to MDS or leukemia.49

Inherited/Congenital Bone Marrow

Failure Syndromes

Compared with acquired aplastic anemia, patients with inher-

ited/congenital bone marrow failure syndromes present at an

earlier age and may have characteristic physical stigmata. The

three most common inherited/congenital bone marrow failure

disorders associated with pancytopenia are Fanconi anemia,

dyskeratosis congenita, and Shwachman-Bodian-Diamond

syndrome.

Fanconi Anemia

Fanconi anemia (FA) is a chromosome instability disorder

characterized by aplastic anemia, physical abnormalities, and

cancer susceptibility. In 1927 Dr. Guido Fanconi first described

this syndrome in three brothers with skin pigmentation, short

stature, and hypogonadism.57 FA has a prevalence of 1 to 5 cases

per million.58 The carrier rate is 1 in 300 in the United States

and Europe, with a threefold higher prevalence in Ashkenazi

Jews and South African Africaners.59 FA is the most common of

the inherited bone marrow failure syndromes.

Clinical findings. Patients with FA have variable features

and symptoms. Physical malformations may be present at birth, though hematologic abnormalities may not appear until older

childhood or adulthood. Furthermore, only two-thirds of

patients have physical malformations.3,58,60 These anomalies

vary considerably, though there is a higher frequency of skeletal

abnormalities (thumb malformations, radial hypoplasia, mi-

crocephaly, hip dislocation, and scoliosis); skin pigmentation

(hyperpigmentation, hypopigmentation, café-au-lait lesions);

short stature; and abnormalities of the eyes, kidneys, and geni-

tals.58-60 Low birth weight and developmental delay are also

common.

The symptoms associated with pancytopenia usually

become apparent at 5 to 10 years of age, though some patients

may not present until adulthood.3,59 Individuals with FA also

have an increased cancer risk. This includes an increased inci-

dence of leukemia in childhood and solid tumors (e.g., oral,

esophageal, anogenital, cervical) in adulthood.61 In approxi-

mately 5% of cases a malignancy is diagnosed before the FA is

recognized.61

Genetics and pathophysiology. Patients with FA typically

have biallelic mutations or deletions in one of at least 21 genes:

FANCA, FANCB, FANCC, FANCD1 (BRCA2), FANCD2,

FANCE, FANCF, FANCG (XRCC9), FANCI, FANCJ (BRIP1/

BACH1), FANCL, FANCM, FANCN (PALB2), FANCO

(RAD51C), FANCP (SLX4), FANCQ (ERCC4 or XPF), FANCR

(RAD51), FANCS (BRCA1), FANCT (UBE2T), FANCU

(XRCC2), and FANCV (MAD2L2, REV7).58,62 The mode of

inheritance is autosomal recessive except for FANCB, which is

X-linked recessive. Mutations in the FANCA gene occur with

the highest frequency (60%); this is followed by FANCC (14%)

and FANCG (10%), whereas mutations in the other FANC

genes are much less common.3,60,63 The relationship between

mutations in the FA genes and disease pathology is not clear.

Cells are highly susceptible to chromosome breakage after

exposure to DNA cross-linking agents. FA cells may also have

accelerated telomere shortening and apoptosis, a late S-phase

cell cycle delay, hypersensitivity to oxidants, and cytokine

dysregulation.3,58,60,64

The range of FA protein function is not completely known,

but these proteins participate in a highly elaborate DNA dam-

age response pathway. The FA pathway consists of a nuclear

core complex, a protein ID complex, and effector proteins.60,64

The FA proteins A, B, C, E, F, G, L, and M form the nuclear core

complex; proteins D2 and I form the ID complex; and the effec-

tor proteins are D1, J, N, O, P, Q, R, S, T, U, and V. 60,62,64 The core

complex facilitates the monoubiquitylation and activation of

the ID complex. The ID complex then localizes with effector

DNA repair proteins at foci of DNA damage to effect DNA

repair.60,64

Laboratory findings. Laboratory results are similar to those

in acquired aplastic anemia, with pancytopenia, reticulocytope-

nia, and a hypocellular bone marrow. Macrocytic RBCs are

often the first detected abnormality, and thrombocytopenia

usually precedes the development of the other cytopenias.

Fetal hemoglobin (Hb F) may be strikingly elevated, and a-

fetoprotein is also increased.

Chromosomal breakage analysis is the diagnostic test for

Fanconi anemia.58 Patients’ peripheral blood lymphocytes are cultured with the DNA cross-linking agents diepoxybutane

(DEB) or mitomycin C (MMC). Compared with normal

lymphocytes, FA cells have a greater number of characteristic

chromosome breaks and ring chromosomes, indicating in-

creased fragility.3,58 Caution must be taken in interpreting

peripheral blood results because they may be negative in the

10% to 15% of FA patients who have somatic mosaicism as a

result of a reversion of one abnormal allele to the normal

type.3,58 Chromosome breakage studies can be performed on

cultured skin fibroblasts from a skin biopsy specimen in these

cases.58,65 Diagnosis is confirmed by genetic testing for FA gene

mutations or deletions.58

Treatment and prognosis. More than 90% of FA patients

develop bone marrow failure by 40 years of age.61 Furthermore,

one-third of patients develop MDS and/or acute myeloid leuke-

mia (AML) by a median age of 14 years, and 25% develop solid

tumors by a median age of 26 years.61,66 Squamous cell carcino-

mas of the head and neck, anogenital region, and skin are the

most common solid tumors, followed by tumors of the liver,

brain, and kidney.61 Patients with FA have an increased risk of

developing vulvar cancer (4300-fold), esophageal cancer (2300-

fold), AML (800-fold), and head/neck cancer (700-fold) com-

pared with the general population.67 Approximately 3% of pa-

tients develop more than one type of malignancy.66 Left

untreated, death by 20 years of age secondary to bone marrow

failure or malignancy is common. Patients with mutations in

the FANCC gene experience bone marrow failure at a particu-

larly young age and have the poorest survival.58,66 Increased

telomere shortening in FA cells is associated with more severe

pancytopenia and a higher risk of malignancy. However, the

precise role of telomere shortening in the evolution of bone

marrow failure and cancer is currently unclear.68

Supportive treatment for cytopenia includes transfusions

and administration of cytokines (G-CSF and GM-CSF).58,59

The only curative treatment is hematopoietic stem cell trans-

plant, preferably from an HLA-identical sibling, although high-

resolution (molecular) HLA-matched unrelated donors can be

used with almost the same success rate.69 It is important to

screen donor siblings for FA with DNA breakage studies and

gene mutation analysis before transplant. Patients should also

have decreased intensity pretransplant conditioning because of

their underlying chromosomal instability.58,66 Gene therapy has

been attempted in clinical trials but has not been successful.

Dyskeratosis Congenita

DC is a rare inherited bone marrow failure syndrome with an

incidence of approximately 4 cases per million per year.58,70

Clinical findings. DC is characterized by mucocutaneous

abnormalities, bone marrow failure, and pancytopenia. The

typical clinical presentation involves a triad of abnormal skin

pigmentation, dystrophic nails, and oral leukoplakia. Skin and

nail findings usually appear before 10 years of age.3,58 By age 30,

80% to 90% of patients have bone marrow abnormalities.3

Patients can also manifest a wide range of multisystem abnor-

malities, including pulmonary fibrosis, liver disease, develop-

mental delay, short stature, microcephaly, prematurely gray hair

or hair loss, immunodeficiency, dental caries, and periodontal disease.58,71 Patients have a 40% risk of cancer by age 50, most

commonly AML, MDS, and epithelial malignancies.70

Genetics and pathophysiology. DC chromosomes have very

short telomeres, and inherited defects in the telomerase com-

plex are implicated in the pathophysiology.71 The telomerase

complex synthesizes telomere repeats to elongate chromosome

ends, maintaining the telomere length needed for cell survival.

Patients with DC typically have mutations in one of at

least 11 genes. The mode of inheritance is X-linked recessive,

autosomal dominant, or autosomal recessive.3,58,71 The best-

characterized form results from one or more mutations on the

long arm of the X-chromosome on the DKC1 gene coding for

dyskerin. Dyskerin is a ribonucleoprotein involved in RNA pro-

cessing, and it associates with TERC (telomerase RNA compo-

nent) in the telomerase complex. Autosomal dominant forms

are mainly caused by mutations in the genes that encode TERC,

TERT (telomerase enzyme), or TINF2 (component of the

shelterin complex that regulates telomere length).58,71 In the

autosomal recessive form, mutations in TERT, RTEL1, ACD,

CTC1, NHP2, NOP10, PARN, and WRAP53 have been identi-

fied.58,71 The proteins encoded by these genes are also involved

in telomere maintenance. Although the exact pathophysiologic

mechanisms are still unknown, the shortened telomeres in DC

cause premature death in the rapidly dividing cells in the bone

marrow and epithelium and likely lead to genomic instability

and a predisposition to cancer.3,58,72

Laboratory findings. Pancytopenia and macrocytic RBCs

are typical peripheral blood findings. The fetal hemoglobin

level may also be increased. About 75% of patients have an

identified mutation in one of the 11 telomerase complex genes

and can be diagnosed by genetic testing.58,71 A flow cytometry

FISH (flow-FISH) test for detection of very short telomeres in

WBC subsets is used as a diagnostic test for those with

suspected DC who lack mutations in known genes.72 Patients

with FA, SBDS, and acquired aplastic anemia may also have

cells with shortened telomeres, though they are not found in

multiple WBC subsets.72 In contrast, DC cells often have short-

ened telomeres in several WBC subsets, including naive T cells

and B cells.72

Treatment and prognosis. Median survival for patients with

DC is 42 years.70 Approximately 60% to 70% of deaths are due

to bone marrow failure complications. Approximately 10% to

15% of deaths result from severe pulmonary disease, and 10%

of deaths result from malignancies.3,58 Treatment with hemato-

poietic stem cell transplantation has not been optimal because

of the high incidence of fatal pulmonary fibrosis and vascular

complications.3 Although androgen therapy produces a tran-

sient response in 50% to 70% of patients, it does not halt the

progression of the bone marrow failure.3

Shwachman-Bodian-Diamond Syndrome

SBDS is a multisystem disorder characterized by pancreatic in-

sufficiency, cytopenia, skeletal abnormalities, and a predisposi-

tion for hematologic malignancies. The incidence has been es-

timated to be approximately 8.5 cases per 1 million live births.58

Clinical findings. Patients with SBDS have peripheral blood

cytopenia and decreased pancreatic enzyme secretion.45 The pancreatic insufficiency causes gastrointestinal malabsorption,

which typically presents in early infancy.3 Patients have neutro-

penia and immune dysfunction and are at increased risk of

severe infections and sepsis.45,73 Nearly all SBDS patients have

delayed bone maturation, and approximately 50% have failure

to thrive and short stature.45,74

Genetics and pathophysiology. SBDS is an autosomal reces-

sive disorder, and 90% of patients have biallelic mutations in

the SBDS gene.3,45,73 The SBDS gene is involved in ribosome

biogenesis and mitotic spindle stability,71,75 but its relationship

to the disease manifestations is currently unknown. There are

quantitative and qualitative deficiencies in CD341 cells, dys-

functional bone marrow stromal cells, increased apoptosis and

mitotic spindle destabilization in hematopoietic cells, and short

telomeres in peripheral blood granulocytes.3,45,73,75

Laboratory findings. Nearly all patients with SBDS have

neutropenia (less than 1.5 3 109 neutrophils/L).76 Half of pa-

tients also develop anemia or thrombocytopenia, and one

fourth develop pancytopenia.76 The RBCs are usually normo-

cytic but can be macrocytic, and approximately two-thirds of

patients have elevated Hb F.45,76 Bone marrow is usually hypo-

cellular but can be normal or even hypercellular. Because of

pancreatic insufficiency, 72-hour fecal fat testing shows in-

creased fat excretion, and serum trypsinogen and isoamylase

levels are decreased compared with age-related reference inter-

vals.73 Compared with cystic fibrosis, which can have a similar

malabsorption presentation, patients with SBDS have normal

sweat chloride tests. Testing for the SBDS gene mutation is

commercially available and should be done in suspected pa-

tients and their parents.

Treatment and prognosis. In some cases no treatment of

hematologic features is needed. However, if needed, treatment

consists of G-CSF for neutropenia, transfusion support for

anemia and thrombocytopenia, and enzyme replacement for

pancreatic insufficiency. The risk of AML and MDS is approxi-

mately 19% at 20 years and 36% at 30 years.77 Allogeneic stem

cell transplantation is recommended in cases of severe pancyto-

penia, AML, or MDS. Unfortunately, despite supportive care

and attempted curative therapy, 5-year overall survival is 60%

to 65%, with many deaths occurring from severe infections and

malignancy.45,73 Poor outcomes after transplant occur as a result

of graft failure, transplant-related toxicities, and recurrent leu-

kemia.

Differential Diagnosis

A distinction must be made between acquired aplastic anemia,

inherited/congenital bone marrow failure syndromes, and other

causes of pancytopenia, including PNH, MDS, megaloblastic

anemia, and leukemia. The importance of a correct diagnosis is

clear because diagnostic conclusions dictate therapeutic man-

agement and prognosis. The distinguishing features of these

conditions are listed in Tables 19.2 and 19.3.

Alternative diagnoses include lymphoma, myelofibrosis, and

mycobacterial infections, which also may present with pancyto-

penia. However, these diagnoses often can be distinguished

with a careful history, physical examination, and laboratory

testing. Review of a peripheral blood film by an experienced morphologist is important. If needed, bone marrow evaluation

and molecular testing for chromosome abnormalities and gene

mutations can further distinguish these diagnoses. Anorexia

nervosa also may present with pancytopenia. In these cases the

bone marrow is hypocellular and has a decreased number of fat

cells.8 The cytopenias revert with correction of the underlying

disease.

OTHER FORMS OF BONE MARROW FAILURE

Pure Red Cell Aplasia

Pure red cell aplasia (PRCA) is a rare disorder of erythropoiesis

characterized by a selective and severe decrease in erythroid

precursors in an otherwise normal bone marrow. Patients have

severe anemia (usually normocytic), reticulocytopenia, and

normal WBC and platelet counts. PRCA may be acquired or

congenital. It is important to distinguish between acquired and

congenital forms because they require different therapeutic

approaches.

Acquired Pure Red Cell Aplasia

Acquired PRCA may occur in children or adults and can be

acute or chronic. Primary PRCA may be idiopathic or autoim-

mune-related. Secondary PRCA may occur in association with

an underlying thymoma, hematologic malignancy, solid tumor,

infection, chronic hemolytic anemia, collagen vascular disease,

or exposure to drugs or chemicals.78,79 Therapy is first directed

at treatment of the underlying condition, but IST may be

considered if the PRCA is not responsive. Cyclosporine is

associated with a higher response rate (65% to 87%) than

corticosteroids (30% to 62%) and is better suited for long-term

maintenance if needed.79

The acquired form of PRCA in young children is also known

as transient erythroblastopenia of childhood (TEC). A history of

viral infection is found in half of patients, which is thought to

trigger an immune mechanism that targets red cell produc-

tion.80 The anemia is typically normocytic, and Hb F and eryth-

rocyte adenosine deaminase levels usually are normal.78,80 Red

cell transfusion support is the mainstay of therapy if the child

is symptomatic from anemia. Normalization of erythropoiesis

occurs within weeks in the vast majority patients.80 There may

be a genetic predisposition to TEC in some families.80

Congenital Pure Red Cell Aplasia: Diamond-Blackfan

Anemia

Diamond-Blackfan anemia (DBA) is a congenital erythroid

hypoplastic disorder of early infancy with an estimated inci-

dence of 7 to 10 cases per million live births.58 Mutations have

been identified in at least 17 genes: those that encode structural

ribosome proteins, RPS7, RPS10, RPS17, RPS19, RPS24, RPS26,

RPS27, RPS28, and RPS29 in the 40S subunit and RPL5, RPL11,

RPL15, RPL26, RPL27, RPL31, and RPL35A in the 60S subunit,

as well as the gene encoding GATA1, a transcription factor im-

portant for hematopoieisis.58,71,81 Mutations in the RPS19 gene

occur with the highest frequency (25%), followed by RPL11 and

RPS26 (approximately 6% each); the other known mutations

are uncommon.71,81,82 An additional 15% to 20% of cases can be

accounted for by haplo-deletions of these same ribosome genes,

but many mutations still remain unidentified.58,83 Nearly 50%

of DBA mutations are linked to an autosomal dominant inheritance pattern, less than 1% with an X-linked recessive

pattern (GATA1), and sporadic mutations have also been re-

ported.58,71,81 Mutations in ribosomal proteins disrupt ribosome

biogenesis in DBA, but the pathophysiologic mechanisms lead-

ing to the clinical manifestations are currently unknown.

More than 90% of patients show signs of the disorder during

the first year of life, with a median age of 8 weeks; however,

some patients with DBA are asymptomatic until adulthood.84

Approximately half of patients have characteristic physical

anomalies, including craniofacial dysmorphisms, short stature,

and neck and thumb malformations.58,84

The characteristic peripheral blood finding is a severe mac-

rocytic anemia with reticulocytopenia.58 The WBC count is

normal or slightly decreased, and the platelet count is normal

or slightly increased. Bone marrow examination distinguishes

DBA from the hypocellular marrow in aplastic anemia, because

there is normal cellularity of myeloid cells and megakaryocytes

and hypoplasia of erythroid cells. The karyotype in DBA is usu-

ally normal, and genetic testing can identify a DBA-related

mutation in about 70% of patients.58 In most cases, Hb F and

erythrocyte adenosine deaminase are increased; these findings

distinguish DBA from TEC, in which these levels are nor-

mal.58,81 Other features distinguishing DBA from TEC are

detailed in Table 19.4.

Therapy includes RBC transfusions and corticosteroids. Al-

though 50% to 75% of patients respond to corticosteroid ther-

apy, side effects can be severe with long-term use, including im-

munosuppression and growth delay.58,81 Overall survival is 75%

at 40 years.84 Bone marrow transplantation improves outcomes,

with greater than 90% overall survival in patients younger than

10 years old transplanted with an HLA-matched related donor,

and 80% in those with a matched unrelated donor.81

Congenital Dyserythropoietic Anemia

The congenital dyserythropoietic anemias (CDAs) are a hetero-

geneous group of rare disorders characterized by refractory

anemia, reticulocytopenia, hypercellular bone marrow with

markedly ineffective erythropoiesis, and distinctive dysplastic

changes in bone marrow erythroblasts. Megaloblastoid devel-

opment occurs in some types, but it is not related to vitamin B12

or folate deficiency. Granulopoiesis and thrombopoiesis are normal. The anemia varies from mild to moderate, even among

affected siblings. Secondary hemosiderosis arises from chronic

intramedullary and extramedullary hemolysis, as well as in-

creased iron absorption associated with ineffective erythropoi-

esis. Iron overload develops even in the absence of blood trans-

fusions. Jaundice, cholelithiasis, and splenomegaly are also

common findings. CDAs do not progress to aplastic anemia or

hematologic malignancies.85

Symptoms of CDA usually occur in childhood or adoles-

cence but may first appear in adulthood.58 CDA is classified into

three major types: CDA I, CDA II, and CDA III. There are rare

variants that do not fall into these categories, and they have

been assigned to four other groups: CDA IV through CDA

VII.58,85 Whether CDA types IV through VII actually are sepa-

rate entities is a matter of some controversy. This merely may be

a reflection of the insensitive tests to classify CDA disorders.

Further gene mutation studies should clarify this issue.

CDA I is inherited in an autosomal recessive pattern and is

characterized by a mild to severe chronic anemia. More than

150 cases have been reported.86 CDA I is caused by mutations

in the CDAN1 or C15orf41 genes.58 CDAN1 (chromosome 15)

encodes codanin-1, a cell-cycle regulated nuclear protein.87,88

The exact role of CDAN1 and C15orf41 mutations in the patho-

physiology of CDA I is unknown. Malformations of fingers or

toes, brown skin pigmentation, and neurologic defects are

found more often in CDA I than in the other CDA subtypes.

The hemoglobin usually ranges from 6.5 g/dL to 11.5 g/dL, with

a mean of 9.5 g/dL.85 RBCs are macrocytic and may exhibit

marked poikilocytosis, basophilic stippling, and Cabot rings.

The erythroblasts are megaloblastoid and characteristically

have internuclear chromatin bridges or nuclear stranding

(Figure 19.3). There are less than 5% binucleated erythroblasts.

The characteristic feature of the CDA I erythroblast is a spongy

heterochromatin with a “Swiss cheese” appearance.85 Treatment

includes IFN-a and iron chelation.58,86

CDA II is the most common subtype and is inherited in an

autosomal recessive pattern. More than 300 cases have been reported.86 It results from mutations in the SEC23B gene on

chromosome 20.89 SEC23B encodes a component of the coat

protein complex (COPII) that forms vesicles for transport of

secretory proteins from the endoplasmic reticulum to the Golgi

apparatus.90 Its exact role in the pathophysiology of CDA II is

unknown. The anemia in CDA II is mild to moderate, with

hemoglobins ranging from 9 g/dL to 12 g/dL and a mean hemo-

globin of 11 g/dL.85 On peripheral blood film, RBCs are normo-

cytic with anisocytosis, poikilocytosis, and basophilic stippling.

The bone marrow has normoblastic erythropoiesis, with 10%

to 35% binucleated forms and rare multinucleated forms.85

Occasional pseudo-Gaucher cells are also evident.85 Circulating

RBCs hemolyze with the Ham acidified serum test but not with

the sucrose hemolysis test.58 For this reason, CDA II is also

known as HEMPAS (hereditary erythroblastic multinuclearity

with positive acidified serum).58 The Ham test is no longer used

for CDA II confirmation, given the difficulty of appropriate

quality control and the relative lack of testing availability

in most laboratories.85 RBCs also show abnormal migration

of band 3 using sodium dodecyl sulfate polyacrylamide gel

electrophoresis.85 Treatment includes splenectomy and iron

chelation.58,86

CDA III is the least common of the CDA subtypes, with

about 60 cases reported in the literature, the majority being

from one Swedish family.91 This familial autosomal dominant

form is associated with mutations in the KIF23 gene, which

codes for a protein involved in cytokinesis.91,92 The nonfamilial

or sporadic form is extremely rare, with fewer than 20 cases

reported.86,91 The anemia is mild, and the hemoglobin is usually

in the range of 8 to 14 g/dL, with a mean of 12 g/dL.85 RBCs are

macrocytic, and poikilocytosis and basophilic stippling are evi-

dent. The bone marrow has megaloblastic changes, and giant

erythroblasts with up to 12 nuclei are a characteristic feature.

Patients rarely require RBC transfusions, and iron overload

does not occur.

Myelophthisic Anemia

Myelophthisic anemia is due to the infiltration of abnormal

cells into the bone marrow and subsequent destruction and

replacement of normal hematopoietic cells. Metastatic solid

tumor cells (particularly from lung, breast, and prostate), fibro-

blasts, and inflammatory cells (such as those found in miliary

tuberculosis and fungal infections) have been implicated.93,94

Cytopenia results from the release of substances such as cyto-

kines and growth factors that suppress hematopoiesis and de-

stroy stem, progenitor, and stromal cells.94 With disruption of

normal bone marrow architecture by the infiltrating cells, the

marrow releases immature hematopoietic cells. Furthermore,

because of the unfavorable bone marrow environment, stem

and progenitor cells migrate to the spleen and liver and estab-

lish extramedullary hematopoietic sites.94 Because blood cell

production in the liver and spleen is inefficient, these extramed-

ullary sites also release immature cells into the circulation.93

The severity of anemia is mild to moderate, with normocytic

erythrocytes and reticulocytopenia. Peripheral blood findings

include teardrop erythrocytes and nucleated RBCs, as well

as immature myeloid cells (leukoerythroblastic blood picture)

Figure 19.4 Myelophthisic Anemia Showing a Leukoerythroblastic

Blood Picture. Note a myelocyte, three orthochromic normoblasts, a

giant platelet with abnormal morphology, a micromegakaryocyte, and

teardrop red blood cells. (Peripheral blood, Wright-Giemsa stain, 31000.)

and megakaryocyte fragments (Figure 19.4).93 The infiltrating

abnormal cells are detected in a bone marrow aspirate or biopsy

specimen.

Anemia of Chronic Kidney Disease

Anemia is a common complication of chronic kidney disease

(CKD), with a positive correlation between anemia and renal

disease severity.95,96 The Centers for Disease Control and Pre-

vention reported that in 2017 approximately 30 million adults

in the United States had CKD.97 The primary cause of anemia

in CKD is inadequate renal production of erythropoietin.95,96

Without erythropoietin, the bone marrow lacks adequate stim-

ulation to produce RBCs. Another contributor to the anemia of

CKD is chronic inflammation. Inflammatory cytokines increase

production of hepcidin by the liver, which decreases the iron

available for erythropoiesis (Chapter 17).96,98 Uremia also in-

hibits erythropoiesis and increases RBC fragility.98,99 Further-

more, hemodialysis and frequent blood draws result in chronic

blood loss. Anemia of CKD is normocytic and normochromic

with reticulocytopenia. Burr cells are a common peripheral

blood film finding in cases complicated by uremia.

Anemia in CKD can lead to cardiovascular complications,

cognitive impairment, and suboptimal quality of life.96 The

2012 Kidney Disease: Improving Global Outcomes (KDIGO)

Clinical Practice Guideline for Anemia recommends periodic

hemoglobin testing in patients with CKD and investigation of

the anemia if the hemoglobin is less than 13.0 g/dL in adult

men and less than 12 g/dL in adult women.95 In anemic adult

CKD patients, a trial of oral iron (non-dialysis patients) or in-

travenous iron (dialysis patients) is recommended when the

transferrin saturation is  30% and serum ferritin level

is  500 ng/mL.95 The Guideline also recommends consider-

ation of therapy with an erythropoiesis-stimulating agent

(ESA) if the hemoglobin falls below 10 g/dL.95 Using ESA

therapy to maintain the hemoglobin at more than 11.5 g/dL is

not recommended because of the increased risk of cardiovascu-

lar complications.95 Successful therapy requires adequate iron tores, so plasma ferritin level and percent transferrin satura-

tion are also periodically monitored.

Patients may become hyporesponsive to ESA therapy

because of functional iron deficiency (FID). In FID the bone

marrow is unable to release iron rapidly enough to accommo-

date the accelerated erythropoiesis. The transferrin saturation

remains less than 20%, but the serum ferritin level is normal

or increased, indicating adequate iron stores.100 Patients with

FID may require intravenous iron therapy to reach or maintain target hemoglobin levels, even with high ESA doses.100

Researchers have proposed diagnostic criteria for FID in

CKD: decreased reticulocyte hemoglobin content and in-

creased soluble transferrin receptor (Chapter 8), and greater

than 10% hypochromic RBCs in the peripheral blood.100,101

Other causes of ESA hyporesponsiveness include chronic in-

flammatory disease, infection, malignancy, aplastic anemia,

antibody-mediated pure red cell aplasia, and some hemoglobin

disorders.