Differential Diagnosis Philip Nivatpumin, Steven Gore

Contents

2.1 Introduction . . . . 5

2.2 Nutritional Disorders . . . . 6

2.3 Congenital . . . . 6

2.3.1 Fanconi Anemia . . . . 6

2.3.2 Congenital Dyserythropoietic Anemia 7 2.3.3 Hereditary Sideroblastic Anemia . . . 7

2.4 Toxic Disorders . . . . 8

2.5 Infectious Disorders . . . . 8

2.6 Other Hematopoietic Disorders . . . . . 8

2.6.1 Idiopathic Aplastic Anemia . . . . 8

2.6.2 Paroxysmal Nocturnal Hemoglobinuria . . . . 9

2.6.3 Myeloproliferative/Myelodysplastic Disorders . . . . 9

2.6.4 Large Granular Lymphocytic Leukemia . . . . 9

2.7Summary . . . . 11

References . . . . 11

Introduction

The myelodysplastic syndromes (MDS) are a heteroge- neous group of clonal hematopoietic stem cell disorders characterized by two main features: ineffective hemato- poiesis and a variable risk of transformation to acute myeloid leukemia (AML) (Steensma and Tefferi 2003).

The term ªmyelodysplastic syndromeº itself emerged in the 1970s amidst controversies surrounding the var- ious presentations of this disorder; subsequent attempts have repeatedly been made to further specify a more precise classification (Anonymous 1976; Greenberg et al. 2000). Although advances in the genomic profiling of various malignancies promise improvement in a clas- sification system for MDS as well, our current schemes still rely on a mixture of clinical, morphologic and cy- togenetic features (Alizadeh et al. 2001; Culligan and Ja- cobs 1992; Miyazato et al. 2001).

Given the lack of a pathologic ªgold standard,º var- ious minimal criteria have been proposed for the diag- nosis of MDS (Culligan and Jacobs 1992; Gardais 2000;

Greenberg et al. 2000; Tricot 1992). They rely on mor- phologic evidence of dysplasia, peripheral cytopenias, karyotypic abnormalities and the presence of increased bone marrow blasts. However, those findings may be nonspecific and related to other pathologic processes, including nutritional, toxic, infectious and other clonal hematopoietic conditions. Bone marrow specimens from normal individuals have been reported to demon- strate mildly dysplastic hematopoiesis (Bain 1996;

Champion et al. 1997).

The diagnosis of MDS should be considered in any patient with unexplained cytopenia(s) or monocytosis.

A careful examination of the peripheral blood smear and bone marrow aspirate is essential to identify char- acteristic morphologic changes in any or all hemato- poietic lineages. In some cases, only the peripheral blood smear shows significant evidence of dysplasia.

The bone marrow in MDS is typically hypercellular with dysplasia in one or more lineages (Rios et al. 1990).

However, a distinct subset of MDS that has a hypocellu-

Philip Nivatpumin, Steven Gore

lar marrow with features mimicking aplastic anemia, pure red cell aplasia, and other bone marrow failure states is well recognized (Garcia-Suarez et al. 1998). As stated, however, these features may be present in a vari- ety of other conditions. A thorough workup of other causes of bone marrow conditions with pathologic fea- tures similar to MDS is mandatory, particularly in the absence of confirmatory clonal cytogenetic abnormali- ties.

2.2 Nutritional Disorders

Nutritional megaloblastic anemias have been described for over 100 years. The hallmark megaloblast results from impaired DNA synthesis as a result of vitamin B12 (cobalamin) or folate deficiency. Animal products (meat and dairy) are the sole dietary source of cobala- min in humans. It takes years to develop deficiency of cobalamin (Green and Kinsella 1995). Antibodies to in- trinsic factor (pernicious anemia) are a common cause in the elderly, and other causes of intestinal malabsorp- tion (e.g., sprue, bacterial overgrowth, etc.) account for remaining cases. Folate is found in animal products and leafy green vegetables. Because folate deficiency may develop within months, decreased dietary consumption accompanied with alcohol abuse is a common etiology.

Macrocytic anemia is the most common presenta- tion of vitamin B12 or folate deficiency. Bone marrow findings show erythroid hyperplasia with an abnormal morphology, as well as megaloblastoid granulopoiesis and megakaryocytopoiesis. These features may mimic either AML or MDS (Green and Kinsella 1995). MDS may present with macrocytosis, reduced reticulocytosis and pancytopenia, making it indistinguishable from these nutritional deficiencies. In addition to other clin- ical features (e.g., cognitive changes, decreased proprio- ception, glossitis), evaluation of the peripheral blood smear is often helpful to distinguish MDS from megalo- blastic anemia. While reduced neutrophil lobulation (i.e., pseudo-Pelger-HuŸt abnormality) and hypogranu- lar neutrophils are more characteristic of the peripheral blood smear of MDS, all of the ªtypicalº changes in megaloblastoid anemias may be seen in MDS. Hyper- segmentation, increased neutrophil lobulation, and megaloblastoid changes have all been reported in MDS. Low serum B12 levels and elevated methylmalonyl CoA levels suggest the diagnosis of megaloblastic ane- mia in vitamin B12 deficiency. Reduced red blood cell

folate and an elevated homocysteine level are seen in fo- late deficiency. Bone marrow cytogenetics and flow cy- tometry studies are normal in B12 and folate deficiency, although one must be aware that MDS and nutritional deficiencies may coexist in some patients (Drabick et al. 2001). Treatment with intramuscular vitamin B12 or oral folate will result in improvement of anemia and resolution of hypersegmentation within several weeks. However, neurologic sequelae recover only over the course of months and, in some cases, may be irre- versible. A ªtherapeutic trialº of B12 is not recom- mended in the absence of documented vitamin deficien- cy.

2.3 Congenital 2.3.1 Fanconi Anemia

In younger patients, congenital causes of bone marrow failure must be considered in the differential diagnosis of MDS. Although most of these disorders present very early in childhood and are associated with other charac- teristic distinguishing clinical features (e.g., anatomic abnormalities), there have been well-described reports of patients with Fanconi anemia (FA) presenting with atypical features later in adolescence and even early adulthood (Butturini et al. 1994; Cavenagh et al. 1996).

Fanconi anemia is an autosomal recessive disease char- acterized by congenital abnormalities, ineffective hema- topoiesis, and an increased risk of developing acute leu- kemias, myelodysplastic syndrome, and certain solid tu- mors (Tischkowitz and Hodgson 2003). A disorder of impaired DNA repair, FA has a heterogeneous clinical presentation with most patients, presenting in child- hood with a combination of skeletal, gastrointestinal, renal, hematologic, and cardiac defects. Patients with FA may present with aplastic anemia, MDS, or acute leukemia. Macrocytosis is often the first hematologic abnormality, followed by thrombocytopenia and neu- tropenia, and, eventually, pancytopenia. In one study of 388 FA patients, the risks of developing hematologic abnormalities and death from hematologic causes were 98% and 81%, respectively (Butturini et al. 1994).

In the presence of typical features, the diagnosis is

easily made by the chromosome breakage test. In

younger patients presenting with a hypocellular bone

marrow and cytopenias, FA should be considered. Treat-

ment is supportive in mild cases. The most definitive

6

treatment is hematopoietic stem cell transplantation with 2-year survival rates as high as 60±70% with hu- man leukocyte antigen (HLA)-identical sibling donors (Gluckman et al. 1995).

2.3.2 Congenital Dyserythropoietic Anemia Congenital dyserythropoietic anemia (CDA) is a rare group of hereditary disorders characterized by ineffec- tive erythropoiesis and distinct morphologic abnormal- ities in bone marrow erythroblasts (Heimpel 2004). The range of genetic heterogeneity and precise molecular defects is beyond the scope of discussion in this chapter.

However, some key features are worth noting in the dif- ferential diagnosis with MDS. Three features are often present: ineffective erythropoiesis, hyperbilirubinemia, and a distinct morphologic appearance of erythroblasts that is easily recognized by trained individuals. The rar- ity of this disease often delays diagnosis, with estimates of up to 60% of cases diagnosed in adulthood, despite multiple prior evaluations for laboratory abnormalities (Greiner et al. 1992; Heimpel 2004). The peripheral blood smear may show anisocytosis, poikilocytosis, ba- sophilic stippling, and occasional erythroblasts. The leading morphologic abnormality is the presence of bi- nucleate erythroblasts in 10±50% of bone marrow eryth- roblasts. Bone marrow cellularity is often increased. In- direct hyperbilirubinemia is often present, and serum haptoglobin is low. Acid lysis testing may be helpful in some subtypes, but diagnosis often relies on expert morphologic analysis, high clinical suspicion, and ge- netic testing (Wickramasinghe 1998, 2000). Treatment is dependent on specific subtypes and may include in- terferon and splenectomy. Prevention of iron overload significantly improves outcome.

2.3.3 Hereditary Sideroblastic Anemia

Hereditary sideroblastic anemias (HSA) are a heteroge- neous group of disorders and may have X-linked or autosomal inheritance or may be associated with spo- radic congenital defects. Clinically, patients generally present with a mild anemia that is stable for many years.

Mild to moderate hepatosplenomegaly is common. With time, patients develop symptoms and signs of chronic iron overload, reflecting the underlying pathophysio- logy of increased iron absorption due to ineffective

erythropoiesis and impaired heme biosynthesis (Bot- tomley 2000; Koc and Harris 1998). Iron overload may become manifest clinically with cirrhosis, cardiac dis- ease, impaired glucose tolerance and diminished libido.

In children, growth retardation may occur.

A number of molecular defects have been identified in the pathophysiology of the hereditary sideroblastic anemias, including defects involving erythroid amino- levulinate synthase (ALAS2), ferrochelatase, cyto- chrome oxidase, thiamine transporter-1 and a variety of mitochondrial proteins (Alcindor and Bridges 2002). In many patients, the cause of sideroblastic ane- mia is unknown. However, the common mechanism in- volves impaired heme biosynthesis and an accumula- tion of mitochondrial iron in erythroblasts.

The complete blood count typically shows a mild anemia. White blood cells and platelets are usually nor- mal. Erythrocyte hypochromia is a consistent finding.

Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) are all low and usually parallel the degree of anemia. Iron overload is reflected in the iron studies. Serum ferritin and transferrin saturation are increased, and serum transferrin levels are de- creased. In X-linked sideroblastic anemia, free erythro- cyte protoporphyrin is low. Bone marrow biopsy shows increased reticuloendothelial iron in bone marrow macrophages. Normoblastic erythroid hyperplasia with ringed sideroblasts is characteristic.

Cytogenetic studies are normal unless a concomi- tant myelodysplasia is present. Depending on the in- heritance pattern, various molecular tests may be used to evaluate for the underlying cause. Mutations in the ALAS2 gene are common in X-linked sideroblastic ane- mia. Mutations in the ABC7 transporter gene and SLC19A2 gene are found in X-linked sideroblastic ane- mia with ataxia and thiamine-responsive megaloblastic anemia syndrome (Alcindor and Bridges 2002; Bottom- ley 2000). A variety of other specific genetic tests may be conducted depending on clinical suspicion.

There is no definitive treatment. Bone marrow

transplantation has been attempted with some success

(Gonzalez et al. 2000; Urban et al. 1992). X-linked side-

roblastic anemia is responsive to pyridoxine (vitamin

B6) treatment in up to two thirds of patients. In con-

trast, true acquired refractory anemia with ringed side-

roblasts is rarely pyridoxine-responsive (May and

Bishop 1998). Treatment of iron overload is critical to

improving survival. Therapeutic phlebotomy and iron chelation are both important mainstays of therapy.

2.4 Toxic Disorders

A variety of toxic exposures may result in bone marrow dysplastic changes that resemble myelodysplasia. A de- tailed clinical history and physical examination is the best way to elucidate these alternative diagnoses. Alco- hol is a frequent cause of macrocytosis and anemia, in- dependent of serum folate levels. Heavy use of alcohol has been reported to cause vacuolization of erythroid and granulocytic precursors (Girard et al. 1987). With severe alcohol abuse, bone marrow hypoplasia and pan- cytopenia may result. Changes are reversible with cessa- tion of alcohol intake.

Acute poisoning with heavy metals (e.g., lead, mer- cury, cadmium, gold, arsenic, etc.) is often suggested by a history of exposure. Chronic exposure, however, may have subtle clinical findings and may present with bone marrow changes, including dysplasia, hypoplasia and aplastic anemia. The diagnosis is made by a careful oc- cupational and exposure history, clinical examination, and a heavy metal screen of the peripheral blood.

Many medications can cause alterations in bone marrow pathology. Certain antibiotics, anticonvulsants, nonsteroidal anti-inflammatory drugs and antithyroid medications may all cause a clinical scenario similar to aplastic anemia. Dysplastic changes in the bone mar- row aspirates of patients recovering from cancer che- motherapy and radiation therapy may be indistinguish- able from MDS.

2.5 Infectious Disorders

Viruses are well-described causes of hematologic abnor- malities. Cytopenias are common. Bone marrow changes include features ranging from dysplasia to out- right aplasia. Hematologic abnormalities are well de- scribed in HIV-positive individuals. Macrocytosis is common either as a direct effect of the virus or a side effect of antibiotic or antiretroviral therapy. A compre- hensive review of 216 bone marrow samples from HIV-positive individuals was reported in 1991 (Karcher and Frost 1991). Megaloblastic hematopoiesis and mye- lodysplasia were seen in 38% and 69% of specimens blindly reviewed, respectively. HIV testing should be

considered in patients with unexplained hematologic and bone marrow abnormalities.

Many other viruses have been associated with aplas- tic bone marrow changes including parvovirus B19 and hepatitis (Brown et al. 1997; Kurtzman and Young 1989).

Parvovirus B19 is a common viral infection causing er- ythema infections in children and an influenza-like ill- ness in adults. In immunosuppressed patients or pa- tients with high red blood cell turnover (e.g., hemolytic anemia), parvovirus infection may result in a pure red cell aplasia and rarely aplastic anemia. Bone marrow biopsy and aspirate may show classic giant pronormo- blasts. The diagnosis is made by serology and polymer- ase chain reaction. Non-A, non-B, and non-C hepatitis have been reported in association with bone marrow failure and may be considered in the differential diagno- sis. Virtually any other severe infection may cause sig- nificant bone marrow abnormalities, but the clinical scenario is usually helpful in establishing the underly- ing diagnosis.

2.6 Other Hematopoietic Disorders 2.6.1 Idiopathic Aplastic Anemia

A variety of other primary hematologic conditions may

have similar features to MDS. Bone marrow specimens

may display hypo- or hypercellularity. Idiopathic aplas-

tic anemia (AA) can be difficult to distinguish from hy-

pocellular MDS. Up to 20% of MDS patients have bone

marrow cellularity that is less than 25% or less than ex-

pected based on age (Nand and Godwin 1988; Tuzuner

et al. 1995). We have already discussed earlier many

other causes of bone marrow failure and bone marrow

hypoplasia/aplasia that may resemble hypocellular

MDS. The etiology of ªidiopathicº AA is thought to

be autoimmune in nature in most patients. This is bol-

stered by clinical observations of improvement with im-

munosuppressive therapy (Young 2002). Bone marrow

immunostaining for CD34 positivity may distinguish

aplastic anemia from MDS (Orazi et al. 1997; Scopes

et al. 1994). Numerous studies have demonstrated that

CD34

bone marrow cells are reduced in specimens

from patients with AA in comparison with patients with

MDS. This is consistent with both the presumed mech-

anism of autoimmune destruction of progenitor cells in

AA and the malignant clonal nature of MDS. Immuno-

8

staining for CD34 combined with cytogenetic analysis help in distinguishing AA and hypocellular MDS.

The clinical responsiveness of a subset of MDS to immunosuppressive therapy suggests an overlap with both aplastic anemia and paroxysmal nocturnal hemo- globinuria (see below). Ultimately, the ªdiagnosisº given to these disorders is of less importance than the under- lying mechanism of bone marrow failure, which may al- low appropriate selection of effective therapy.

2.6.2 Paroxysmal Nocturnal Hemoglobinuria Paroxysmal nocturnal hemoglobinuria (PNH) is an- other clonal hematopoietic disorder to consider in the differential diagnosis. It is characterized by a defect in the glycosylphosphatidylinositol (GPI)-anchor due to mutations in the PIG-A gene (Rosse 1997). The loss of many proteins bound to the GPI-anchor on the surface of hematopoietic cells is thought to lead to the clinical manifestations of hemolysis, venous thrombosis, and bone marrow failure. Furthermore, given its clonal na- ture, it has also been described in the setting of MDS, myeloproliferative disorders, and the progression to AML (Longo et al. 1994; Nakahata et al. 1993). The diag- nosis of PNH should be considered in any patient pre- senting with cytopenias and a hypocellular bone mar- row. Classically diagnosed by the sucrose lysis and Ham's test, it may now be identified by the absence of GPI-linked proteins, CD55 and CD59, on the surface of peripheral blood cells by using monoclonal antibodies and flow cytometry. Flow cytometric quantification of GPI-anchor binding using fluorescent-labeled inactive toxin aerolysin (FLAER) appears to be the most sensi- tive method for PNH diagnosis (Brodsky et al. 2000).

Treatments include supportive care, immunosuppres- sion, and hematopoietic stem cell transplantation.

2.6.3 Myeloproliferative/Myelodysplastic Disorders

There can be overlap between some clinical and patho- logic features of MDS and certain myeloproliferative disorders. Indeed, chronic myelomonocytic leukemia (CMML) was previously included in the French-Ameri- can-British (FAB) classification of MDS due to the dys- plastic features seen on bone marrow biopsy. However, in many ways, CMML shows classic features of a myelo-

proliferative disorder. The World Health Organization (WHO) recognized this in their recent recommendation that CMML, atypical chronic myelogenous leukemia (CML), and juvenile myelomonocytic leukemia (JMML) be removed from their prior CML and MDS categories and placed in a separate category of ªMDS/MPDº (Har- ris et al. 1999). Patients present with dysplastic bone marrow features and increased white blood cell counts.

Karyotypic or molecular analysis to detect the Philadel- phia chromosome or the BCR/ABL fusion gene easily identifies typical CML. However, some patients display features of both CML and CMML, yet lack the Philadel- phia chromosome or the BCR/ABL fusion gene (Bennett et al. 1994). These patients with ªatypical CMLº show marrow dysplastic changes similar to MDS yet may have progressive leukocytosis and organomegaly reminiscent of myeloproliferative disorders. These patients have a significantly worse prognosis than typical CML patients, with a reported median survival of 24 months (Onida et al. 2002).

Bone marrow fibrosis is another pathologic entity that is nonspecific and may cloud the diagnosis of MDS. Although mild to moderate degrees of bone mar- row fibrosis have been reported in up to 50% of patients with MDS, diffuse fibrosis is rare (Steensma et al. 2001;

Sultan et al. 1981). MDS with myelofibrosis may be dis- tinguished from myelofibrosis with myeloid metaplasia (MMM), often on clinical grounds. Patients with the former are often pancytopenic with trilineage dysplasia and have atypical megakaryocytic proliferation. MMM typically has splenomegaly and extramedullary hemato- poiesis. Furthermore, MMM is often more indolent.

While the distinction with MMM may be easier, it may be difficult to distinguish MDS with myelofibrosis from acute myelofibrosis, the accelerated phase of chronic myelogenous leukemia, and acute megakaryo- cytic leukemia.

2.6.4 Large Granular Lymphocytic Leukemia

Large granular lymphocytic (LGL) leukemia may also

present with pancytopenia and bone marrow hypopla-

sia. It is a form of indolent non-Hodgkin lymphoma,

characterized by a clonal proliferation of T cells or nat-

ural killer cells. The diagnosis is often suggested by

findings of chronic neutropenia, a modest absolute lym-

phocytosis, and the presence of morphologically typical

cells in the peripheral blood. Anemia and thrombocyto-

10

Table 2.1. Differential diagnosis of myelodysplasia

Diagnoses Clinical features Smear/pathology Testing

Vitamin B12 deficiency

Altered mental status, paresthesias, Glossitis, decreased propriocep- tion, Elderly, history of malabsorption

Hypersegmented neutrophils, macrocytosis, hypercellular mar- row

Serum B12 low, MMA high, elevated LDH and bilirubin

Folate deficiency Elderly, history of alcohol use, or depression

No neurologic symptoms

Similar to B12 deficiency Elevated homocysteine level, decreased RBC folate

Fanconi anemia Autosomal recessive, presents in childhood or early adulthood, other family members. Involved, other hematologic or solid malig- nancy. Usually with anatomic de- fects

Macrocytosis, cytopenias, hypocellular marrow

Chromosomal breakage test

Congenital dyserythropoietic anemia

Hyperbilirubinemia, hemolysis, tiredness, multiple prior evalua- tions without diagnosis

Anisocytosis, poikilocytosis, baso- philic stippling, circulating eryth- roblasts, bone. Marrow with binucleate erythroblasts

Acid lysis test Direct genetic testing

Hereditary

sideroblastic anemia

Low MCV, low MCH, low MCHC, elevated ferritin, low transferrin, iron overload, Pyridoxine respon- sive in some cases

Hypochromic anemia, increased marrow iron, ringed sideroblasts

Free erythrocyte proto- porphyrin. Gene muta- tion analysis

Heavy metal exposure

Occupational history, mental status changes

GI symptoms

Pancytopenia and hypocellular bone marrow

Heavy metal screen.

Testing of coworkers/

T family HIV Risk factors, opportunistic infec-

tions. Virtually any other organ system may be affected

Any cytopenia may be seen, macrocytosis autoimmune throm- bocytopenia, bone marrow with megaloblastosis and dysplasia

HIV testing; CD4 count;

viral load

Parvovirus B19 Influenza-like illness, rash, arthral- gias/arthritis. Erythema infectio- sum in children,

immunosuppressed and patients with hemolytic anemia most prone

Typically anemia, may resemble pure red cell aplasia, bone marrow with giant pronormoblasts

Parvovirus IgM/IgG titers

PCR for Parovirus B19

Idiopathic aplastic anemia

Present with symptoms related to cytopenias

Pancytopenia, hypocellular bone marrow

Rule out other causes CD34

percentage of marrow Precursors (usually low in AA) Paroxysmal nocturnal

hemoglobinuria

Thrombosis, Budd-Chiari presentation, hemolysis

May present with any cytopenia or pancytopenia. Bone marrow ranges from normal to similar to aplastic anemia; may progress to MDS or AML. May arise from prior treatment of AA

CD55, CD59 of peri-

pheral blood

penia are not uncommon. Macrocytosis has been re- ported in over 20% of cases (Dhodapkar et al. 1994).

However, in some patients these peripheral blood find- ings may be subtle, and patients may present with pan- cytopenia alone. In a Mayo clinic series of 203 patients with T-cell LGL leukemia, 14% presented with pancyto- penia at initial presentation, and nine patients fulfilled criteria for aplastic anemia (Dhodapkar et al. 1994; Go et al. 2000). LGL leukemia has been described in asso- ciation with rheumatoid arthritis and other hematologic disorders, including monoclonal gammopathy of unde- termined significance and multiple myeloma (Loughran et al. 1988). Evaluation of the peripheral blood smear of- ten shows the presence of LGLs. LGLs are large, typically contain azurophilic granules, and have an abundant cy- toplasm and a round nucleus. Bone marrow pathology in the majority of cases shows clonal lymphocytic infil- trates. A minority of patients presents with a hypocellu- lar bone marrow resembling aplastic anemia or hypo- cellular MDS. Diagnosis is confirmed by demonstration of a clonal population of T cells or natural killer (NK) cells either in the bone marrow or peripheral blood.

Prognosis is generally excellent, with some reports of median survivals in excess of 10 years (Dhodapkar et al. 1994). Treatment consists of immunosuppressive therapy or low-dose cytotoxic therapy and is usually re- served for symptomatic patients with progressive dis- ease (Loughran 1993).

2.7 Summary

The differential diagnosis of MDS is broad (Table 2.1).

Many conditions resemble myelodysplasia in one or more features. In the absence of cytogenetic abnormal-

ities, a thorough evaluation of congenital, nutritional, toxic, infectious, and other clonal disorders is necessary.

Based on the results of initial laboratory testing and careful evaluation of the peripheral blood smear and bone marrow, a focused number of additional tests of- ten yield the diagnosis (Table 2.2).

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Diagnoses Clinical features Smear/pathology Testing

Atypical CML Splenomegaly, extramedullary infiltrates. Poor response to therapy, progression to AML

Leukocytosis, absence of baso- philia, bone. Marrow hyper- cellular with erythroid dysplasia

BCR-ABL negative

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