bone marrow

1536 n HematolympHoid SyStem

1536 n

Bone MarrowdaNiel a. aRBeR

C H a p t e R

43

SPECIMEN HANDLING

NORMAL BONE MARROW AND AN APPROACH TO BONE MARROW EVALUATION

NEOPLASTIC AND PROLIFERATIVE PROCESSESAncillary Techniques Useful in Bone Marrow EvaluationMyelodysplastic Syndromes and Acute LeukemiaMyeloproliferative NeoplasmsMyelodysplastic/Myeloproliferative NeoplasmsPlasma Cell Disorders

Lymphoid ProliferationsGranulomatous and Histiocytic DisordersMast Cell DiseaseMetastatic Tumors

APPROACH TO THE NON-NEOPLASTIC BONE MARROWAplasiasHyperplasiasOther Non-neoplastic Marrow Changes

BONE MARROW REPORT

The bone marrow biopsy specimen differs from biopsy material of most other organs because a proper evaluation of the bone marrow requires the incorporation of a variety of specimen types to arrive at an accurate and complete diagnosis. Bone marrow studies should be evaluated in con-junction with clinical data, review of peripheral blood smears, and complete blood count data, as well as with bone marrow aspirate smears or imprints. Many cases also benefit from immunophenotyping or molecular genetic and cyto-genetic studies. The results of all these studies should be considered in making a final diagnosis. This chapter empha-sizes this multifactorial approach to bone marrow evalua-tion and will attempt to highlight the specimen types that require the use of ancillary techniques for accurate diagno-sis. Complete clinical information is essential for proper triage of material for microbiology cultures, flow cytometry, and cytogenetic or molecular genetic studies because these methods usually require fresh tissue.

SPECIMEN HANDLING

Most bone marrow specimens are sternal aspirates or iliac crest aspirates with or without biopsies. Details of the pro-cedure and of specimen preparation are well described else-where.1-4 Aspirate smears are preferably made at the bedside but may also be made in the laboratory after the procedure. Bone marrow aspirate material may be immediately placed into “purple-top” ethylenediamine tetra-acetic acid (EDTA)–containing tubes for the preparation of smears at a later time. This method limits the clotting of the aspirate speci-men and allows for material to be submitted for ancillary studies or even particle sections. Whether the smears are made at the bedside or from EDTA tubes, the aspirate should

be grossly evaluated for the presence of bone marrow par-ticles. Such particles should be removed and placed on a slide for the actual smear preparation. The absence of par-ticles on a smear limits diagnostic usefulness in many cases, and such smears often show changes of peripheral blood. Samples from many pediatric patients, however, do not have gross particles in the aspirate material despite numerous bone marrow elements in the specimen.

Similar to the preparation of peripheral blood smears,5 only a small amount of bone marrow material should be gently smeared across the slide. Different methods may be employed, including the use of coverslips to smear the particles gently. An alternate method is to place a small drop of particulate marrow toward the labeled end of a slide.2 The outer portion of the drop, away from the label, is then touched with the edge of a second slide and the marrow is gently “pulled” across the original slide with the edge of the second slide. Large drops of marrow on a slide result in thick and bloody smears that are difficult to interpret. Smears made with too much pressure distort or destroy the marrow cells. Multiple air-dried smears should be prepared, although the actual number depends on the number of marrow particles in the specimen and the clinical indication for the procedure. Although touch preparations or imprints are suggested on all specimens with a bone marrow biopsy, they are essential in cases with a “dry tap” in which the aspirated material has the appearance of peripheral blood.6

Representative aspirate smears and imprints should be stained with a Romanowsky stain. The actual stain type varies among laboratories and includes Giemsa, Wright-Giemsa, and May-Grünwald-Giemsa stains. Rapid review of these smears is helpful in determining the need for ancillary studies, such as cytochemistry, immunophenotyping, and cytogenetic and molecular genetic methods.C

BoNe maRRow n 1537

Clot biopsy sections are often made from coagulated aspirate material. This material contains predominantly blood as well as small marrow particles that can be embed-ded and processed for sections stained with hematoxylin-eosin (H&E) or periodic acid–Schiff (PAS). Despite the absence of clotting of EDTA-anticoagulated aspirate speci-mens, the bone marrow particles that are left after making smears can be filtered and embedded for histologic evalua-tion.7 This method provides a more concentrated collection of bone marrow particles, but it may not yield any material in some pediatric patients.

Trephine core biopsy is not performed in all patients. However, this procedure is essential in the evaluation of patients suspected of having disease that may only focally involve the bone marrow, such as malignant lymphoma, or for tumor staging, and it is preferred in all patients. Bilateral bone marrow biopsies are recommended for patients under-going bone marrow staging.8 Imprint slides from the biopsy specimens may be made either at the bedside or in the labo-ratory. To make these slides in the laboratory, the bone marrow core should be submitted fresh, on saline-damp-ened gauze, with the imprints made immediately to allow for adequate fixation of the biopsy specimen. Otherwise, the imprints should be made at the bedside and the biopsy can be submitted in fixative. Many laboratories prefer mer-curic chloride–based fixatives for bone marrow specimens, but distribution of this substance to hospital wards and offices is often not advisable and heavy metal fixatives usually render the sample unsuitable for molecular genetic studies. Formalin and Bouin’s fixative are also commonly used. After fixation, the core biopsy is decalcified and pro-cessed for H&E-stained or PAS-stained sections.

NORMAL BONE MARROW AND AN APPROACH TO BONE MARROW EVALUATION

It is often easiest to evaluate a bone marrow specimen by comparing the specimen with what would be expected in the normal bone marrow.9,10 The initial evaluation on low magnification of the biopsy specimen is of the marrow cel-lularity. Estimates of cellularity on aspirate material are described,11 but they may be unreliable in variably cellular bone marrow.12 The normal cellularity varies with age and evaluation of cellularity must always be made in the context of the patient’s age.13 In children, the bone marrow is approximately 80% cellular through the age of 10 years and then slowly declines in cellularity until 30 years of age, where it remains at approximately 50% cellularity. Bone marrow cellularity declines again in elderly patients to approximately 30% at 70 years. Because of the variation in cellularity by age, the report should clearly indicate whether the stated cellularity in a given specimen is normocellular, hypocellular, or hypercellular.

Estimates of cellularity may be inappropriately lowered by several factors. Subcortical bone marrow is normally hypocellular and these areas should be ignored in the cel-lularity estimate. In addition, technical artifacts may cause false lowering of marrow cellularity determinations. Tears in the section caused during processing and cutting as well

as artifactual displacement of marrow from bony trabeculae should not be considered in the estimate.

After bone marrow cellularity has been evaluated, the cellular elements must be considered. The three main bone marrow cell lines, maturing granulocytes, erythroid precur-sors and megakaryocytes, should be evaluated first. Matur-ing granulocytes are the most common cell type in the normal marrow with a 2 : 1 to 3 : 1 granulocyte-to-erythroid ratio; the higher ratio is more common in women.10 All stages of granulocyte and erythroid maturation are normally present, with blast cells usually representing less than 3%. The various stages of maturation are best evaluated on the aspirate smear material, but the location of immature cell clusters is most precisely identified on the core biopsy. Immature clusters of myeloid and erythroid cells normally occur adjacent to bony trabeculae. Megakaryocytes are easily identifiable on smear and biopsy material in the normal marrow and should consist of predominantly mature forms with hypersegmented nuclei.

Lymphocytes normally represent 10% to 15% of cells on aspirate smears, but lymphoid precursors and mature lymphocytes may be normally increased in children and elderly patients, respectively. The lymphoid precursors, or hematogones,14 are maturing B cells and do not aggregate on the biopsy material of children, despite their presence on aspirate smears. Lymphoid aggregates, however, are common on biopsy material of elderly patients and are non-paratrabecular in location. These cells are predomi-nantly T lymphocytes. Cells that are present at a lower fre-quency in the bone marrow include monocytes, plasma cells, mast cells, eosinophils, basophils, and osteoblasts. These cells normally represent less than 5% of marrow cells on smears.10

Cells and proliferations that do not normally occur in the marrow, including histiocyte accumulations or granulo-mas, fibrosis, serous atrophy, and neoplastic cells, should be systematically assessed in all specimens. The bony tra-beculae should also be evaluated for evidence of osteopenia, osteoblastic proliferations, and the changes of Paget’s disease. Iron stores of the normal marrow are predomi-nantly in histiocytes within marrow particles, but red blood cell iron incorporation is normally seen on oil immersion in scattered cells that usually have one or two siderotic granules adjacent to the nucleus.

NEOPLASTIC AND PROLIFERATIVE PROCESSES

Ancillary Techniques Useful in Bone Marrow Evaluation

Ancillary techniques are essential for the proper diagnosis of many bone marrow neoplasms. Despite the advances in immunophenotyping and cancer genetics, morphologic evaluation still remains the initial step in the bone marrow study and morphologic changes can reliably suggest further studies to identify clinically significant immunophenotypic and genetic findings in some cases. These morphologic features are discussed in the context of specific diseases, as are specific findings of the various ancillary tests. The C


general utility and applications of ancillary testing, however, are discussed later.

Cytochemistry

Despite the widespread use of immunophenotyping in the diagnosis of hematopoietic neoplasms, cytochemical studies are still of importance with some classification systems.15 This is particularly true of the acute leukemias; however, a large menu of cytochemical tests is probably not necessary for most leukemias. Figure 43-1 provides a practical algo-rithm of diagnostic cytochemical tests for acute leukemia classification. However, many leukemia types require immu-nophenotyping studies for precise classification, and the routine use of cytochemistry in acute leukemia is becoming less common. Myeloperoxidase or Sudan black B staining, by cytochemical means, remains the hallmark of a cyto-chemical diagnosis of acute myeloid leukemia (AML) in most cases. Some cases, such as minimally differentiated AML or monoblastic leukemias, are myeloperoxidase nega-tive. The use of a nonspecific esterase cytochemical tech-nique, such as α-naphthyl butyrate esterase, is still the most specific means of identifying monocytic differentiation for classification purposes and can be useful in both acute leu-kemia evaluations and in the workup of chronic myelo-monocytic leukemia (CMML). Cytochemistry is of limited value in the diagnosis of acute lymphoblastic leukemia (ALL). Although negative results of peroxidase cytochemi-cal studies are expected in ALL, they do not sufficiently exclude myeloid leukemia and should not be used as the sole evidence of lymphoid lineage. PAS staining, frequently showing “chunky” positivity in lymphoblast cytoplasm, is also not sufficiently specific to be used diagnostically.

The Prussian blue stain for iron is a histochemical stain that is commonly employed on bone marrow specimens.16 Although it may be performed on clot or biopsy material, it is most reliable and useful on bone marrow aspirate smears as long as sufficient particles are present on the

smear.17 Iron staining is useful in identifying reticuloendo-thelial iron stores in the evaluation of a patient for iron deficiency or overload and is also helpful in the evaluation of red blood cell iron incorporation. Iron stores are often graded from 0 to 6+,18 as summarized in Table 43-1, and such grading correlates well with other chemical measures of iron. The identification of an increase in iron within erythroid precursors, particularly in the form of ringed sid-eroblasts, is helpful in the diagnosis of myelodysplasia and AML with myelodysplasia-related changes. The other cyto-chemical test that is used on bone marrow specimens is the detection of tartrate-resistant acid phosphatase (TRAP) in hairy cell leukemia (HCL),19 although this cytochemical study has been largely replaced by immunophenotypic studies.

Immunophenotyping

Immunophenotyping studies are essential for the proper diagnosis of lymphoblastic malignant diseases and are helpful in the classification of mature lymphoid neoplasms and some myeloid neoplasms. In addition, these studies can provide a characteristic immunologic “fingerprint” of acute leukemia that may be useful in the subsequent evaluation of residual disease.

flow cytometry

Some antibodies that are useful in the immunophenotypic evaluation of blastic proliferations by flow cytometry are listed in Table 43-2. The markers most useful for lympho-mas are discussed in Chapter 41. Several consensus reports and reviews regarding the use of flow cytometry immuno-phenotyping in hematologic malignant disesases20-26 offer useful guidelines on the use of this methodology on periph-eral blood and bone marrow specimens. Flow cytometry primarily detects surface antigens, although some cytoplas-mic and nuclear antigens may also be detected. Flow cytom-etry has the advantage of allowing for the evaluation of several thousand cells in a rapid manner and the ability to assess the expression of multiple antigens on a single cell. In addition, the use of CD45 versus side scatter gating strategies allows for only cells with specific characteristics (e.g., blast cells or lymphoid cells) to be evaluated and this method greatly increases the ability to detect residual disease in a specimen.27

Figure 43-1 n Algorithm of the cytochemical evaluation of acute leuke-mia. A percentage of at least 30% bone marrow blasts is used for the French-American-British Cooperative Group (FAB) classification, but newer classifications require only 20% or more blasts.

Bone marrow blasts ≥30% (20%)Myeloperoxidase or Sudan black B

+(≥3%) –(<3%)

�-Naphthylbutyrateesterase

�-Naphthylbutyrateesterase

M1, M2,most M3,

M6a

Some M3,M4

M5 M4 M0†, M6b,M7†, ALL†

–*(<20%) –*(<20%)+(20%–80%)† +(20%–80%)+(>80%)

*Staining of less than 20% of cells or punctate perinuclear staining of any percentage of cells is considered a negative result for �-naphthyl butyrate esterase.†These cases require immunophenotyping for diagnosis.

taBle 43-1Histologic Grading of Iron Storage in Bone Marrow Aspirate Material

0/Negative No iron identified under oil immersion1+ Small iron positive particle only visible under oil immersion2+ Small, sparsely distributed iron particles usually visible

under low magnification3+ Numerous small particles present in histiocytes throughout

the marrow particles4+ larger particles throughout the marrow with tendency to

aggregate into clumps5+ dense, large clumps of iron throughout the marrow6+ Very large deposits of iron, both intracellular and

extracellular, that obscure cellular detail in the marrow particles

data from Gale e, torrance J, Bothwell t: the quantitative estimation of total iron stores in human bone marrow. J Clin invest 42:1076-1082, 1963.

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BoNe maRRow n 1539

immunohistochemistry

Immunohistochemical analysis of paraffin sections of the clot or core biopsy is ideal for the assessment of focal lesions that may not be available, because of sampling differences or dry taps, on aspirate material submitted for flow cytom-etry. This would include focal bone marrow involvement by malignant lymphoma. This method is also useful for the characterization of tumors that are not routinely assessed by flow cytometry, such as metastatic carcinomas and small round cell tumors of childhood. Because of limitations in the detection of some antigens by paraffin section immuno-histochemistry, flow cytometry immunophenotyping is pre-ferred for the evaluation of lymphoproliferative disorders and acute leukemias. When such material is not available, immunohistochemistry may still provide diagnostic infor-mation.28-30 Useful antibodies for the evaluation of lympho-matous proliferations are discussed in more detail in Chapter 41. Paraffin section antibodies useful for the evaluation of acute leukemia proliferations are given in Table 43-3. As with all immunophenotyping studies, pertinent positive and negative findings should be obtained with a panel of antibodies because the detection of a single antigen is hardly ever sufficiently lineage specific. For example, whereas ter-minal deoxynucleotidyl transferase (TdT) is usually detect-able in lymphoblastic malignancies, it is also present in a subgroup of myeloid leukemias.

Molecular Genetic and Cytogenetic Analysis

Cytogenetic and molecular genetic studies on bone marrow specimens offer valuable information in certain clinical situ-ations, and the prognostic significance of karyotypic changes in acute leukemia is well established. In general, routine karyotype analysis is the preferred first-line test in newly

diagnosed leukemias and when myelodysplasia is suspected because a multitude of acquired genetic abnormalities may be detected by this method. When cryptic or masked trans-locations are suspected, when a precise genetic breakpoint with prognostic implications must be confirmed, or when residual disease testing is needed, molecular genetic tests are useful. This testing is also helpful in identifying gene rearrangements in lymphomas that are not readily identifi-able by karyotype analysis and to detect some lymphoma translocations that may not be consistently detected by karyotype analysis. Details about the specific molecular genetic abnormalities associated with bone marrow diseases are discussed in the context of those diseases.

Large numbers of cytogenetic abnormalities occur with acute leukemias and some of them are summarized in Tables 43-4 and 43-5. The most common groups of abnormalities are those that disrupt transcription factors, the tyrosine kinase translocations, retinoic acid translocations, and 11q23 abnormalities.31,32 Translocations that involve genes

taBle 43-2Selected Useful Flow Cytometry Markers in Acute Leukemia

General

Cd45

Myeloid

Cd11cCd13Cd15Cd33Cd65Cd117Cytoplasmic myeloperoxidase

Myelomonocytic

Cd14Cd64

Megakaryocyte

Cd41Cd61

Plasma Cell

Cd38Cd138

Immature B Lineage

Cd10Cd19Cd22tdtCd79a

Mature B Lineage

Cd19Cd20Cd79aκ/λ

T Lineage

Cd2Cd5Cd7Cd4/Cd8tdtSurface and cytoplasmic Cd3

Others

Cd34Cd56Hla-dR

Hla, human leukocyte antigen; tdt, terminal deoxynucleotidyl transferase.

taBle 43-3Selected Useful Paraffin Section Antibodies in Acute Leukemia

Immature B-Cell

Cd10Cd79atdt

T-Cell

Cd2Cd3Cd5tdt

Myeloid

myeloperoxidaseCd163

Others

Cd34Hla-dR

Hla, human leukocyte antigen; tdt, terminal deoxynucleotidyl transferase.

taBle 43-4Some Common Cytogenetic and Molecular Genetic Abnormalities in Acute Myeloid Leukemia and Myelodysplasia

Translocations Involved GenesMost Common Disease Type

inv(3)/t(3;3)(q21;q26) RPN1/EVI1 myelodysplasiat(3;21)(q26;q22) EVI1, EAP, or MDS1/RUNX1 myelodysplasiat(3;5)(q25;q34) NPM1/MLF1 myelodysplasia,

m2, m6t(8;21)(q22;q22) RUNX1/RUNX1T1 m2t(6;9)(p23;q34) DEK/NUP214 m1, m2, m4t(7;11)(p15;p15) NUP98/HOXA9 m2, m4t(15;17)(q22;q21) PML/RARA m3t(11;17)(q23;q21) ZBTB16/RARA m3t(11;17)(q13;q21) NuMA/RARA m3t(5;17)(q31;q21) NPM1/RARA m3inv(16)/t(16;16)(p13;q22) CBFB/MYH11 m4eot(9;11)(p22;q23) AF9/MLLT3 m5other 11q23

abnormalitiesMLL m4, m5

t(1;22)(p13;q31) RBM15-MKL1 m7

Mutations FLT3 amlNPM1 amlCKIT aml

aml, acute myeloid leukemia. C


that encode transcription factor proteins are some of the most common in leukemia. Of these, the core binding factor, a transcription factor involved in normal hematopoiesis, is one of the best described.33 The core binding factor is formed by an aggregate of different proteins that include the AML1 protein, encoded by the RUNX1/AML1 gene on chro-mosome band 21(q22), and the CBFβ protein, which is encoded on chromosome band 16(q22). Disruption of either one of these chromosome regions, as seen in t(8;21), t(3;21), t(12;21), inv(16), and t(16;16), is associated with development of acute leukemia. The leukemia type that results from each abnormality, however, varies. Despite the great variability in the morphologic and immunopheno-typic features of these leukemias (discussed later in this chapter), they all have a similar molecular genetic mecha-nism that is at least in part related to the development of the leukemic process.

Translocations resulting in the development of a tyrosine kinase fusion protein are a second group of abnormalities in leukemias and include the BCR/ABL1 fusion of t(9;22)(q32;q11) seen in chronic myelogenous leukemia (CML) and some cases of AML, the ETV6/PDGFRB of t(5;12)(q33;p13) in some cases of CMML, and the ETV6/ABL1 fusion of t(9;12)(q32;p13) of some acute and chronic leukemias. These tyrosine kinase proteins interact with other proteins to activate the RAS signaling pathway, which may result in abnormal myeloid proliferation.

The t(15;17)(q22;q11.2) is the most common retinoic acid translocation and is present in most acute promyelo-cytic leukemias. This translocation results in a PML/RARA fusion. Other less common translocations also occur in acute promyelocytic leukemia and involve the RARA gene. Fusion proteins involving RARA negatively inhibit the poorly understood normal functions of the RARA gene and apparently result in the development of acute leukemia. This mechanism of leukemia transformation is unique to the acute promyelocytic leukemias. All-trans retinoic acid overrides the negative inhibitory effect of the leukemic

fusion protein and thus makes it a unique therapeutic agent for this disease.

Translocations involving chromosome band 11q23 are common and at least 50 different translocation partners may occur with this chromosomal region in acute leukemia. Most translocations involve the MLL gene, also known as ALL1 and HRX.34,35 The MLL gene is believed to function as a homeotic transcription regulator, but the way in which translocations involving this gene cause leukemia is not well understood. The type of leukemia differs with the dif-ferent MLL translocations. Translocations involving the MLL gene also occur in therapy-related acute leukemias and are common following chemotherapy with topoisomerase II inhibitors.

The lymphoid malignant diseases also have evidence of immunoglobulin or T-cell receptor gene rearrangements that may be useful markers of clonality. Such tests are usually not necessary to make a diagnosis of malignancy in cases of acute leukemia, and because lineage infidelity is common in ALLs, these tests may not be useful in defining cell lineage. A unique gene rearrangement in ALL, however, may be sequenced and patient-specific polymerase chain reaction (PCR) primers and probes have been shown to be useful for the monitoring of residual disease. These methods, however, are not available in most medical centers for routine use. The mature lymphoid malignant diseases also demonstrate gene rearrangements and many are associated with unique cytogenetic translocations. These abnormali-ties are discussed in more detail in the context of each disease and in Chapter 41.

Cytogenetic studies are essential to identify the abnor-mality associated with a given leukemia, although t(12;21) and some MLL translocations may be missed by routine karyotype analysis. PCR methods are useful to confirm the precise translocation site and are potentially useful to follow patients for residual disease. Some translocations, including t(8;21) and possibly inv(16), may persist in very low numbers after treatment. Detection of these abnormalities by routine PCR methods does not necessarily predict relapse. For this reason quantitative PCR methods may be of more value for the detection of residual disease after therapy.36 Cytogenetic studies do not detect gene mutations, such as FLT and NPM1 mutations, which are common in some acute leukemia types, and PCR methods are most commonly used to detect these abnormalities.

Myelodysplastic Syndromes and Acute Leukemia

In the past, pathologists and hematologists used the classi-fication systems for acute leukemia and myelodysplastic syndromes (MDSs) developed by the French-American-British (FAB) cooperative group.37-41 These systems are summarized in Tables 43-6 and 43-7. These schemes, however, do not include some of the clinically significant genetic findings of these diseases that were described more recently. In addition, many of the categories of disease are not considered of clinical relevance with modern therapeu-tic approaches. The World Health Organization (WHO) classification of hematopoietic tumors includes both myelo-

taBle 43-5Some Common Cytogenetic and Molecular Genetic Abnormalities in Acute Lymphoblastic Leukemia

Translocation Involved Genes

Precursor B-Cell ALL t(9;22)(q34;q11) BCR/ABL1t(12;21)(p13;q22) ETV6/RUNX1t(1;19)(q23;p13) TCF3/PBXt(17;19)(q22;p13) TCF3/HLFt(4;11)(q21;q23) AFF1/MLLother 11q23

abnormalitiesMLL

Burkitt’s Leukemia t(8;14)(q24;q32) IGH/MYCt(2;8)(p12;q24) IGK/MYCt(8;22)(q24;q11) Ig/MYC

Precursor T-Cell ALL 1q32 abnormalities TAL1t(8;14)(q24;q11) TCRA/MYCt(11;14)(p15;q11) TCRD/LMO1t(11;14)(p13;q11) TCRD/LMO2t(10;14)(q24;q11) TCRD/TLX1del 9(p21) p16 and p15t(1;7)(p34;q34) TCRB/LCK

all, acute lymphoblastic leukemia.

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BoNe maRRow n 1541

dysplasia and acute leukemia and this is now the preferred classification system for these tumors.42 The WHO catego-ries that relate to bone marrow disease are given in Tables 43-8 and 43-9. The classification, especially for AML, is complicated. The Realistic Pathologic Classification (RPC)43 of AML, summarized in Table 43-10, is a conceptual clas-sification that includes the categories of disease that can be realistically diagnosed before cytogenetic results are com-pletely known. This system allows for the addition of cyto-genetic and molecular genetic results that may provide clinically important information. Both the WHO and RPC classifications change the percentage of blast cells required for a diagnosis of AML to 20% or greater for most disease categories and remove any blast cell requirement for cases with the more common recurring cytogenetic abnormali-ties. The first change reflects findings of similar outcomes for patients with myelodysplasia with high blast cell counts when compared with patients with AML who are treated in a similar fashion.44,45 Details of the WHO classification system, its FAB counterparts, and other entities described in the literature are addressed later.

Myelodysplasia

Myelodysplasia, previously termed preleukemia, is a clonal neoplastic proliferation of the bone marrow that may be primary or may follow toxic exposures or therapy.38,46 Primary myelodysplasia is relatively common in elderly patients, is associated with persistent cytopenias, and fre-quently progresses to bone marrow failure with resulting infections or to AML.

Evaluation of the peripheral blood smear is just as impor-tant as evaluation of the bone marrow in patients with sus-pected myelodysplasia, and features of dysplasia are identified in the peripheral blood of most patients with this disease.47 These patients have significant cytopenias, which almost always include anemia and elevation of the red blood cell distribution width (RDW). The anemia is often macro-cytic. The red blood cells frequently have a dimorphic appearance with macrocytes and hypochromic teardrop-shaped cells. The white blood cell count is often low, with or without circulating blast cells. The neutrophils often demonstrate abnormalities of nuclear lobation and abnor-mal cytoplasmic granulation. The nuclei are frequently mature, with the chromatin of a segmented neutrophil, but fail to demonstrate normal segmentation. Abnormal bilobed neutrophil nuclei with mature and clumped nuclear chro-matin and only a thin band of nucleoplasm connecting the lobes are termed pseudo–Pelger-Huet cells. Neutrophils may also show loss of normal cytoplasmic granulation with zonal, hypogranular areas with a “washed-out” appearance and some may have abnormal cytoplasmic vacuoles. Plate-lets are often decreased and many show loss of normal platelet granules.

Similar dysplastic changes are evident in the bone marrow (Fig. 43-2). The bone marrow is usually hypercellular but may be normocellular or even hypocellular. The erythroid series is usually hyperplastic and left shifted with abnor-mally large, megaloblastic erythroid cells with smudged or thick, ropy nuclear chromatin and dark basophilic cyto-plasm with or without cytoplasmic vacuoles. Nuclear-to-cytoplasmic asynchrony is also common as well as bilobated or multilobated erythroid precursors and cells with irregu-lar or lobated nuclear contours. Siderotic granules may be evident even on Romanowsky-stained smears and Howell-

taBle 43-6French-American-British Cooperative Group Classification of Myelodysplastic Syndromes

Peripheral Blood Monocytosis of >1 × 109/L

Peripheral Blood Blasts Bone Marrow Blasts

Ringed Sideroblasts >15% of Erythroid

Cells

Refractory anemia (Ra) − <1% and <5% −Refractory anemia with ringed sideroblasts (RaRS) − <1% and <5% +Refractory anemia with excess blasts (RaeB) − >1% but <5% or ≥5% but ≤20% ±Refractory anemia with excess blasts in

transformation (RaeB-t)− ≥5% or >20% but <30% (or

auer Rod +)†±

Chronic myelomonocytic leukemia (Cmml) + <5% and ≤20% ±Chronic myelomonocytic leukemia in

transformation (Cmml-t)*+ ≥5% or >20% but <30% ±

*Not included in the original French-american-British Cooperative Group (FaB) classification, but generally accepted as a category.†the presence of auer rods with any blast cell count is considered evidence of RaeB-t.

taBle 43-7French-American-British Cooperative Group Classification of Acute Leukemia

Acute Myeloid Leukemia

m0 minimally differentiated acute myeloid leukemiam1 myeloblastic leukemia without maturationm2 myeloblastic leukemia with maturationm3 promyelocytic leukemiam3v microgranular variantm4 myelomonocytic leukemiam4eo myelomonocytic leukemia with eosinophilsm5a monoblastic leukemia (poorly differentiated)m5b monocytic leukemia (differentiated)m6 erythroleukemiam7 megakaryoblastic leukemia

Acute Lymphoblastic Leukemia

l1l2l3 (Burkitt’s)

data from references 37 and 39 through 51.

adapted from Bennett Jm, Catovsky d, daniel mt, et al: proposals for the classification of the myelodysplastic syndromes. Br J Haematol 51: 189-199, 1982.

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taBle 43-8World Health Organization Classification of Primary Bone Marrow Disorders

Myeloproliferative Neoplasms

Chronic myelogenous leukemia, BCR-ABL+

Chronic neutrophilic leukemiaChronic eosinophilic leukemia/hypereosinophilic syndromeChronic idiopathic myelofibrosispolycythemia veraessential thrombocythemiaChronic myeloproliferative neoplasm, unclassified

Myelodysplastic/Myeloproliferative Neoplasms

Chronic myelomonocytic leukemiaatypical chronic myeloid leukemia, BCR-ABL1−

Juvenile myelomonocytic leukemiamyelodysplastic/myeloproliferative neoplasm, unclassifiable

Myelodysplastic Syndromes

Refractory anemiaRefractory anemia with ringed sideroblastsRefractory cytopenia with multilineage dysplasiaRefractory anemia with excess blastsmyelodyplastic syndrome associated with isolated del(5q) chromosome

abnormalitymyelodysplastic syndrome, unclassified


Acute Myeloid Leukemias with recurrent Cytogenetic Abnormalities

acute myeloid leukemia with t(8;21)(q22;q22) (RUNX1/RUNX1T1)acute promyelocytic leukemia; acute myeloid leukemia with

t(15;17)(q22;q12), (PML/RARA) and variantsacute myeloid leukemia with inv(16)(p13q22) or t(16;16)(p13;q22)

(CBFB/MYH11)acute myeloid leukemia with t(9;11)(p22;q23), (MLLT3-MLL)

Acute Myeloid Leukemia with Myelodysplasia-related Changes

acute myeloid leukemia with t(6;9)(p23;q34), (DEK-NUP214)acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2),

(RPN1-EVI1)acute myeloid leukemia (megakaryoblastic) with t(1;22)(p13;q13),

(RBM15-MKL1)therapy-related myeloid neoplasmstopoisomerase ii inhibitor related

Acute Myeloid Leukemia Not Otherwise Specified

acute myeloid leukemia, minimally differentiatedacute myeloid leukemia, without maturationacute myeloid leukemia, with maturationacute myelomonocytic leukemiaacute monoblastic and monocytic leukemiaacute erythroid leukemiaacute megakaryocytic leukemiaacute basophilic leukemiaacute panmyelosis with myelofibrosis

Myeloid Sarcoma

Acute Leukemias of Ambiguous Lineage

Precursor B- and T-Cell Neoplasms

B-lymphoblastic leukemia/lymphomat-lymphoblastic lymphoma/leukemia

Burkitt’s Lymphoma/Leukemia (included in mature B-cell neoplasm category)

taBle 43-9World Health Organization Classification of Myelodysplasia*

Peripheral Blood Blasts Bone Marrow Blasts

Prominent Multilineage Dysplasia

Ringed Sideroblasts

>15% of Erythroid Cells

Refractory cytopenia with unilineage dysplasia <1% and <5% − −Refractory anemia with ringed sideroblasts 0% and <5% − +myelodysplastic syndrome with isolated del(5q) <5% and <5% −† −Refractory cytopenia with multilineage dysplasia <1% and <5% + ±Refractory anemia with excess blasts, type 1 (RaeB-1) <5% or 5%-9% + ±Refractory anemia with excess blasts, type 2 (RaeB-2) ≥5%-19% or 10%-19%(or auer Rod +)‡ + ±

*Cases with a bone marrow blast cell count of >20% are considered acute leukemia in this system. treatment-related myelodysplasia may fulfill criteria for any of the categories but is considered a separate category based on the poor prognosis of patients with a history of previous therapy. Chronic myelomonocytic leukemia is classified as a myelodysplastic/myeloproliferative disease in this system.

†Small, hypolobated megakaryocytes are present. mild dyserythropoiesis may also be seen.‡the presence of auer rods with any blast cell count is considered evidence of RaeB-2 in this system.

Jolly–like nuclear fragments may be present in the cyto-plasm. The granulocyte series may show changes similar to those described in the blood. In addition, myeloblasts are often increased to 3% or more. Some blasts may have cyto-plasmic granules in a more uneven distribution than pro-myelocytes and these cells should be counted as blasts rather than promyelocytes when they are the prominent immature cell type. Megakaryocyte dysplasia is also common in the bone marrow. The most specific findings are nuclear

hyperchromasia and hypolobation, but hyperlobated mega-karyocyte nuclei with detached nuclear segments may also be identified.

A morphologic feature associated with myelodysplasia on biopsy specimens is the detection of aggregates of imma-ture cells away from the bony trabeculae.48 Normal foci of regeneration, in patients who are not hematopoietic stem cell transplant recipients, are located adjacent to bone. Such aggregates of immature cells are termed ALIPs for abnormal

adapted from Swerdlow SH, Campo e, Harris Nl, et al (eds): wHo Classification of tumours of Haematopoietic and lymphoid tissues. lyon, France, iaRC press, 2008.

adapted from Swerdlow SH, Campo e, Harris Nl, et al (eds): wHo Classification of tumours of Haematopoeitic and lymphoid tissues. lyon, France, iaRC press, 2008.

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BoNe maRRow n 1543

localized immature precursors. Detection of such aggregates is associated with a significantly poor prognosis.49 Bone marrow fibrosis is not usually found in association with myelodysplasia, but rare cases occur that may be confused with myeloproliferative disorders and are associated with a worse prognosis.49-52

Refractory anemia (RA) and RA with ringed sideroblasts (RARS) cause persistent anemia that may be macrocytic, but may be morphologically subtle. The characteristic features of dysplasia mentioned earlier may not be obvious in the blood or bone marrow. These patients have erythroid hyper-plasia and often demonstrate mild dyserythropoiesis without other evidence of dysplasia. Blast cells are often not increased and comprise less than 5% of the bone marrow and 1% or less of the peripheral blood.38 Iron staining of an aspirate smear may reveal an increase in red blood cell iron incor-poration and more than 15% of erythroid precursors are ringed sideroblasts (ringing around at least one third of the nucleus) in cases of RARS (Fig. 43-3). In the absence of ringed sideroblasts, a descriptive diagnosis with a request for cytogenetic studies may be indicated before a definite diagnosis of myelodysplasia can be made. The detection of characteristic clonal cytogenetic abnormalities, such 5q or

20q deletions, monosomy 7, trisomy 8, or complex cytoge-netic abnormalities, is diagnostic of RA in this setting.53-55 In the absence of a clonal cytogenetic abnormality, the per-sistence of anemia with the exclusion of other causes may be sufficient for an eventual clinical diagnosis of RA. RA and RARS are the most indolent of the MDSs.

Anemias with increases in ringed sideroblasts (sidero-blastic anemias) are not all clonal MDSs. Hereditary, con-genital, and acquired sideroblastic anemias may occur and a fairly complex classification system of these disorders has been proposed.56 Ringed sideroblasts usually result from a mitochondrial disorder or a defect in normal heme synthe-sis. Acquired causes of ringed sideroblasts include MDSs, but these cells also result from direct toxic exposure to chlor-amphenicol, isoniazid, lead, copper, ethanol, and cycloser-ine.57-59 Of the hereditary sideroblastic anemias, X-linked sideroblastic anemia is one of the best studied. This disease primarily affects male patients and causes microcytic hypo-chromic anemia with ringed sideroblasts that may be con-fused with hemoglobinopathy. Many of these patients carry a point mutation in the δ-aminolevulinate synthase-2 gene (ALAS-2) on chromosome band region X(p11). Distinguish-ing these disorders from true myelodysplasias may be diffi-

taBle 43-10Realistic Pathologic Classification of Acute Myeloid Leukemia

Acute Myeloid Leukemia, de Novo

acute myeloid leukemia with changes suggestive of t(8;21)(q22;q22)acute promyelocytic leukemiaacute myeloid leukemia with abnormal eosinophils suggestive of

inv(16)(p13q22)/t(16;16)(p13;q22)acute megakaryoblastic leukemiaacute myeloid leukemia, not otherwise specified*

Acute Myeloid Leukemia with Multilineage Dysplasia

Acute Myeloid Leukemia, Therapy-related

*Cases found to have an 11q23 abnormality should have reports amended to indicate the prognostic significance of this type of acute myeloid leukemia.

A B

Figure 43-2 n Myelodysplasia. A, The bone marrow aspirate demonstrates dyserythropoiesis with nuclear-to-cytoplasmic asynchrony, abnormally seg-mented neutrophils, and scattered blast cells. B, The bone marrow biopsy may contain aggregates of immature-appearing cells that are not adjacent to bone trabeculae, often referred to as abnormal localized immature precursors (ALIPs).

Figure 43-3 n An iron stain of a bone marrow aspirate smear showing numerous ringed sideroblasts in a case of refractory anemia with ringed sideroblasts (RARS).

data from arber da: Realistic pathologic classification of acute myeloid leukemias. am J Clin pathol 115:552-560, 2001; and arber da, Stein aS, Carter NH, et al: prognostic impact of acute myeloid leukemia classification: importance of detection of recurring cytogenetic abnormalities and multilineage dysplasia on survival. am J Clin pathol 119:672-680, 2003.

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cult, but these conditions should be suspected in children or young adult patients because RARS is extremely unusual in those age groups. Patients with anemia and ringed sid-eroblasts, but no evidence of granulocyte or megakaryocyte dysplasia, often have no cytogenetic abnormalities on routine karyotype analysis and are less likely to progress to myelodysplasia with increased blasts or to acute leukemia.60 Cases with prominent multilineage dysplasia or an increase in blast cells are best classified as one of the other subtypes of myelodysplasia described later.

One morphologically and clinically distinct subtype of RA is the 5q− (5q minus) syndrome.61-63 This syndrome most frequently occurs in elderly women who present with mac-rocytic anemia and normal platelet counts. The bone marrow of these patients has less than 5% blast cells and usually no obvious dysplastic changes of the granulocyte series. Mild dyserythropoiesis is common, but the marked erythroid hyperplasia of other myelodysplasias is infre-quent. The most striking feature of these patients is the presence of increased numbers of abnormal megakaryocytes (Fig. 43-4). More than 50% of megakaryocytes of these patients have monolobated or bilobated nuclei. These mor-phologic findings, when seen in conjunction with a deletion of chromosome band 5(q13q33) as the sole cytogenetic abnormality, are associated with a good prognosis with a low risk of transformation to acute leukemia. Patients with an increase in blast cells or with karyotype abnormalities in addition to 5q− do not share this good prognosis and should not be considered to have the 5q− syndrome. Ringed sid-eroblasts are also uncommon in this syndrome and their presence usually correlates with additional karyotypic abnormalities.

Refractory cytopenia with multilineage dysplasia64,65 with or without ringed sideroblasts was previously included in the categories of RA and RARS or called unclassified myelo-dysplasia. The presence of prominent multilineage dysplasia (two or more cell lines), even without an increase in blast cells, is associated with more severe cytopenias and more aggressive behavior than in RA or RARS. The median sur-vival (24 months in one study) is intermediate between that of RA and RA with excess basts (RAEB).64 Therefore, separa-

tion of these cases into a distinct subtype of myelodysplasia appears warranted.

RAEB is associated with anemia and trilineage dysplasia but also demonstrates an increase in myeloblasts. In the FAB classification, blast cells represent 5% or more (but <20%) of bone marrow nucleated cells or more than 1% (but <5%) of peripheral blood white cells. Although abnor-malities of red blood cell incorporation of iron, including the presence of ringed sideroblasts, are frequently present, this finding does not affect the classification of MDSs with increased numbers of blast cells. The WHO classification further subdivides RAEB into type 1 and type 2. RAEB-1 includes patients with less than 10% bone marrow and less than 5% peripheral blood blasts. RAEB-2 includes patients with 10% or more blasts, but less than 20% blood or bone marrow blasts. Patients with a blast count of less than 10% but with Auer rods are also considered to have RAEB-2 (see later).

RAEB in transformation (RAEB-T) to acute leukemia is defined in the FAB classification as the presence of 20% or more blasts (but <30%) in the bone marrow or the presence of 5% or more blasts in the peripheral blood. Because the WHO classification considers cases with 20% or more peripheral blood or bone marrow blasts to represent AML and considers cases with up to 20% peripheral blood blasts to be RAEB-2, a category of RAEB-T is not part of the WHO classification. The presence of Auer rods in blast cells of either the peripheral blood or bone marrow is also consid-ered evidence of RAEB-T in the FAB classification, even if the overall number of blast cells is not increased, and RAEB-2 in the WHO classification. This criterion is somewhat controversial because some studies have found no correla-tion between the presence of Auer rods and aggressive behavior of myelodysplasia,66 and others seem to confirm that these cases are more aggressive.67 Because of the pos-sible difference in the behavior of cases diagnosed as RAEB-T based solely on the presence of Auer rods, I recommend that the criteria for the diagnosis of RAEB-T or RAEB-2, if it is solely the result of Auer rods, be clearly stated in the report.

MDSs with 17p abnormalities are reported to have distinct morphologic features, but they have variable numbers of blast cells and may fit into any of the previously described categories of MDS.68 They are associated with p53 muta-tions, which are otherwise uncommon in MDSs.69 Myelo-dysplasias with these abnormalities have frequent pseudo–Pelger-Huet cells, abnormal monolobated neutro-phils, and neutrophils with vacuolated cytoplasm. These features, however, are nonspecific and these cases are not a distinct category in the WHO classification.

Some investigators have considered hypocellular MDS to be a distinct entity.70,71 A definition of an MDS with a cel-lularity of less than 30% as hypocellular myelodysplasia is somewhat arbitrary and may not represent a truly hypocel-lular marrow in very elderly patients. For this reason, some investigators have advocated the use of age-specific cutoffs for this diagnosis.70 Other than the low cellularity of the bone marrow of these patients, the morphologic features allow for classification into the categories listed earlier, and I recommend use of the foregoing categories with a comment that the marrow is hypocellular rather than making a non-specific diagnosis of hypocellular myelodysplasia.

Figure 43-4 n The 5q minus syndrome. Increased numbers of small, monolobated megakaryocytes are present on the aspirate smear.C

BoNe maRRow n 1545

Therapy-related MDSs, in the past, were placed into FAB categories as described earlier, but these diseases are usually more rapidly progressive and should be considered a dis-tinct type of MDS that may be independent of the blast cell count.72-77 The WHO classification combines this type of myelodysplasia with therapy-related AML but no longer subdivides them by the previously administered therapeutic agent. MDS/AML following treatment with topoisomerase II inhibitors is most often associated with balanced translocations involving chromosome bands 11(q23) and 21(q22),78 respective sites of the MLL and RUNX1/AML1 genes. However, other more typical de novo leukemia translocations may occur in these patients. The time interval between treatment and disease is relatively short; MDS/AML occurs in 2 to 3 years. Many patients treated with topoisomerase II inhibitors progress directly to AML without the obvious dysplastic changes seen in AML/MDS following alkylating agents. MDS/AML following treatment with alkylating agents has a longer latent period of 7 or more years and is associated with chromosome 5 or 7 deletions or unbalanced 11(q23) and 21(q22) rearrange-ments. Therapy-related myelodysplasia with 17p deletions, with morphologic features as described earlier and p53 mutations, has also been reported following alkylating agent therapy for lymphoma and hydroxyurea for essential throm-bocythemia (ET).79,80

Cytogenetic studies are among the most important ancil-lary studies to be performed in the evaluation of MDSs.81 In cases of morphologically subtle RA, the detection of a char-acteristic clonal karyotypic abnormality, such as monosomy 7, 5q deletions, and trisomy 8, clearly establishes the diag-nosis. The type of cytogenetic abnormality also has prog-nostic significance. Several prognostic scoring systems for myelodysplasia have been proposed, some of which include cytogenetic studies.82-84 The International Prognostic Scoring System for myelodysplasia84 is now widely used. Rather than the disease categories described earlier, this system uses a combination of the peripheral blood changes, blast cell count, and cytogenetic findings to place patients into prognostic categories. This system is summarized in Table 43-11.

Cytogenetic studies therefore are essential in the proper characterization of MDSs, and traditional karyotyping remains the standard for initial assessment. Fluorescence in situ hybridization (FISH) studies, however, are ideal for following these patients once the abnormality is identified. Immunophenotyping studies may be of some value in quan-titation of blast cells. The detection of increased numbers of CD34+ blast cells by flow cytometry or immunohisto-

chemistry,49,85,86 or the detection of aberrant immunopheno-types in the various cell populations,87,88 may complement the morphologic evaluation of these cases.


AML is defined, using FAB criteria,39 as a proliferation of myeloblasts that represent 30% or more of nucleated marrow cells or 30% or more of nonerythroid precursor cells if ery-throid precursors represent 50% or more of marrow nucle-ated cells (FAB M6). The WHO classification of AML lowers the number of blast cells required to diagnose acute leuke-mia to 20% in most cases and has no blast cell requirement for the AMLs with some recurring cytogenetic abnormali-ties. These changes would result in a diagnosis of AML in patients previously considered to have RAEB-T in the FAB classification.

The FAB classification does not incorporate the presence of associated dysplastic changes that are commonly seen in adult patients with AML and includes disease categories of questionable clinical significance.45 Many de novo cases of AML are associated with well-defined cytogenetic transloca-tions that have prognostic significance. The frequency of AML in adults increases with age and may have abnormali-ties similar to the de novo AMLs of childhood or, more commonly, have complex cytogenetic abnormalities that are more suggestive of myelodysplasia. The latter cases also frequently have associated multilineage dysplastic changes in the non–blast cell populations. AML therefore may be divided into two broad categories (see Table 43-10): de novo AMLs, which can be subdivided into different types; and AML with multilineage dysplasia. Various types of AML are described later in contrast to the more limited categories of the RPC of Table 43-10.43 It remains useful to understand the morphologic variants described in the FAB classification and to understand the lack of specificity of some of those variants. The primary criticisms of the FAB classification of AML have focused on the inability to incorporate cytoge-netic changes or to separate cases with dysplastic features from those without, and groups, in addition to the WHO, have tried to incorporate those features into AML classifica-tions.43,89-92 Of the ancillary studies in AML, detection of cytogenetic abnormalities that define prognostically signifi-cant disease groups is probably the most important.45,93-96 Cytochemical evaluation should be a routine part of the diagnostic workup of the AMLs if the FAB classification is being reported, with the addition of immunophenotyping studies to identify cases of minimally differentiated AMLs. Although immunophenotyping is not essential for the diag-

taBle 43-11International Prognostic Scoring System for Myelodysplasia*

Variable/Score 0 0.5 1.0 1.5 2.0

Bone marrow blasts <5% 5%-10% 11%-20% 21%-30%Karyotype Normal, del(5q) or del(20q) all other abnormalities Complex (≥3 abnormalities) or

chromosome 7 abnormalitiesCytopenias 0 or 1 2 or 3

*Risk groups for survival or acute myeloid leukemia evolution are as follows: low (score 0), intermediate-1 (score 0.5-1.0), intermediate-2 (score 1.5-2.0), and high (score ≥2.5).

data from Greenberg p, Cox C, leBeau m, et al: international scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 89:2079-2088, 1997. C


nosis of AML in many cases, it is beneficial in identifying certain disease groups (described later) and in identifying aberrant blast cell immunophenotypes that can be used to monitor follow-up specimens for minimal residual disease.

acute myeloid leukemia, not otherwise specified (who) and related fab categories

The WHO classification includes a large category of AMLs that are termed not otherwise specified. Despite this designa-tion, this disease group includes numerous disease catego-ries, many of which have significant overlap with specific FAB disease groups. Other than the difference in blast cell count needed for a diagnosis of AML mentioned previously, the FAB and WHO categories differ in a few other ways. The subgroups of AML, not otherwise specified do not allow for the presence of prior therapy; the most common recurring cytogenetic abnormalities, or the presence of myelodysplasia-related changes (see later). If any of these features is present, the cases are classified into a different group.

Minimally differentiated acute myeloid leukemia is termed M0 in the FAB classification.41,97-99 The blasts do not dem-onstrate Auer rods or cytoplasmic granules and lack evidence of myeloid or monocytic differentiation by cyto-chemical analysis (Fig. 43-5). Therefore, they test negative for myeloperoxidase, Sudan black B, or nonspecific esterase on cytochemical analysis. Detection of myeloperoxidase expression by immunophenotypic studies is acceptable for this diagnosis. Results of testing for TdT are frequently positive in these cases, and this finding coupled with the negative cytochemical results may lead to an incorrect diagnosis of ALL if adequate immunophenotyping studies are not performed. Detection of myeloid-associated anti-gens, such as CD13, CD33, CD65, and CD117, in the absence of lymphoid specific markers, such as CD3, CD19, CD20, and CD79a, is critical for making the correct diag-nosis of a myeloid lineage leukemia. Other less restrictive criteria for the diagnosis of AML-M0 have been reported that allow for nonspecific esterase positivity or expression of one or more lymphoid antigens. Such cases often have

complex cytogenetic abnormalities or karyotypes sugges-tive of associated MDSs and often demonstrate morphologic evidence of myelodysplasia. If these features are present, a case of this type would not fall under the WHO category of AML, not otherwise categorized, but would be considered AML with myelodysplasia-related changes (see later).

AMLs without or with maturation are termed M1 and M2 leukemias, respectively, in the FAB system. M2 leukemias are distinguished by the presence of maturation to the pro-myelocyte level of differentiation in at least 10% of cells. The blast cells are otherwise similar in the two groups, with the exception of the t(8;21) AMLs discussed later, and include cells with indented nuclei and large nucleoli. The blast cell cytoplasm may contain varying numbers of cyto-plasmic granules, and Auer rods may be seen in either leu-kemia type. Blast cells are usually defined as having 20 or fewer cytoplasmic granules. Some blasts, however, may have more granules and should be considered blast cells if they retain the immature nuclear features of other more typical blasts, rather than the more mature nucleus of a normal promyelocyte. These cells also do not demonstrate the characteristic perinuclear clearing of granules, or hof, of normal promyelocytes. By definition, the presence of 3% or more of the blast cells is positive for myeloperoxidase, and the presence of less than 20% of cells is positive for nonspecific esterase testing on cytochemical analysis.

Differentiating M1 from M2 leukemias is of little signifi-cance in most cases, except for the identification of leuke-mias that may have the cytogenetic abnormality t(8;21)(q22;q22).93-95 Cases with this translocation are a spe-cific subtype of the WHO classification and are discussed in more detail later. Acute myelomonocytic and acute mono-blastic leukemias (M4 and M5) in the FAB classification have cytochemical evidence of monocytic origin by cytochemical analysis using nonspecific esterase stains. These stains include α-naphthyl butyrate esterase and α-naphthyl acetate esterase. The acetate esterase reaction is inhibited by sodium fluoride in monocytic cells. The designation of M4 in the FAB classification is given when 20% to 80% of cells are nonspecific esterase positive and M5 is diagnosed when more than 80% of cells test positive (Fig. 43-6). Subpopula-tions of cells are also myeloperoxidase positive, especially in M4, but some monoblastic leukemias have entirely nega-tive results for myeloperoxidase and Sudan black B testing. Monoblastic leukemias may be further subdivided into those without maturation with immature cells with abun-dant cytoplasm and round immature nuclei, M5a, and those with monocytic maturation with folded nuclei, designated M5b. Immature monocytic cells, including promonocytes that demonstrate immature nuclear chromatin with nucle-oli but have nuclear folds with or without cytoplasmic vacuoles of mature monocytes, are counted as blast cells in these types of leukemias. More pronounced maturation may be evident in the peripheral blood of patients with acute monocytic leukemias, a finding that renders subtyping of these leukemias on blood specimens unreliable. The periph-eral blood finding may suggest CMML, and such a diagnosis should not be made in the blood without excluding acute leukemia by bone marrow examination.

Some patients with acute monoblastic leukemia present with extramedullary myeloid tumors, and gingival infil-trates by monoblasts are classically associated with this type

Figure 43-5 n Minimally differentiated acute myeloid leukemia (M0). The bone marrow shows blasts with scant, basophilic cytoplasm. Immunophe-notyping studies are needed to confirm the myeloid lineage of the cells.C

BoNe maRRow n 1547

of AML.100,101 Despite these clinical presentations, great overlap exists between these leukemias and other AMLs, and monocytic leukemias do not appear to have a worse prognosis when compared with other AML subtypes.45,102 AMLs with monocytic features and abnormal eosinophils (FAB M4Eo) are a unique subgroup and are described with the other AMLs with recurrent cytogenetic abnormalities.

Acute erythroid leukemia is designated M6 in the FAB classification and has many features in common with the MDSs (Fig. 43-7). At least two types of erythroid leukemia are described.39,103,104 The first form is the one described by the FAB. These leukemias have 50% or more bone marrow erythroid precursors. Myeloblasts represent 30% or more of the nonerythroid cell elements of the marrow. These blasts are otherwise similar to the myeloblasts of the other AMLs. This type of AML is also referred to as M6a by some inves-tigators. The WHO category of erythroid leukemia uses similar diagnostic criteria, except myeloblasts, as a percent-age of the nonerythroid cells of the marrow, must only be 20% or more. The second type of erythroid leukemia has been termed pure erythroid leukemia, erythremic myelosis, Di

Guglielmo’s disease, or M6b. This is a proliferation of imma-ture cells with erythroid features104 and some of these cases were probably classified as myelodysplasias in the past. The immature cells have basophilic cytoplasm similar to the erythroid pronormoblast and often have cytoplasmic vacu-oles. The WHO further defines this category of erythroid leukemia as having more than 80% erythroid precursors in the marrow. Although dysplastic erythroid changes are common in both types of erythroid leukemia, they are most striking in M6b. In general, primarily morphologic features make the diagnoses of M6a and M6b. Complex cytogenetic abnormalities, including those common to MDSs, are common in acute erythroleukemia.105,106 For this reason, cases with more than 20% total myeloblasts in the bone marrow may be better classified as AML with myelodysplasia-related changes (see later), and one could argue that the remaining cases actually represent a type of myelodysplasia.43

Acute megakaryoblastic leukemia (FAB M7) is a type of leukemia of immature cells with megakaryocyte features that occurs in children or adults (Fig. 43-8).40,107-109 This

Figure 43-6 n Acute monoblastic leukemia (M5b). The blasts have round to slightly indented nuclei with abundant vacuolated cytoplasm. More prominent nuclear folds are present in some cases.

Figure 43-7 n Acute erythroid leukemia (M6). The bone marrow contains a spectrum of abnormal cells that include dysplastic erythroid precursors with nuclear cytoplasmic asynchrony, red cell precursors with siderotic granules, and immature cells with vacuolated, basophilic cytoplasm.

Figure 43-8 n Acute megakaryoblastic leukemia (M7). A, The aspirate smears are often acellular but may contain blast cells with friable cytoplasm similar to a mature megakaryocyte. This feature is not sufficiently specific for the diagnosis, and a megakaryocytic immunophenotype should be demonstrated. B, The biopsy specimen is usually fibrotic with immature cells and abnormal megakaryocytes.

A B

C


disease group probably represents more than one biologic entity,106 but the blast cell features are similar among them. Maturing megakaryocytes are often easy to identify admixed with the immature cell population. The blasts have fairly uniform, fine nuclear chromatin with varying amounts of basophilic cytoplasm. The cytoplasm often forms blebs or pseudopods, but this feature alone is not sufficient for a diagnosis of M7. Marrow fibrosis, often resulting in an inability to aspirate the marrow, is common. When blast cells are available for evaluation, they test negative for myeloperoxidase and Sudan black B, but may test positive for acid phosphatase and α-naphthyl acetate esterase (sodium fluoride sensitive). Results of immunophenotyping studies are positive for CD13 and CD33 in some cases. Detection of platelet/megakaryocyte-associated antigens, such as CD41, CD42, and CD61, or the detection of platelet peroxidase by electron microscopy110 is essential for the diagnosis. In my experience, detection of at least two mega-karyocytic markers is needed to make a diagnosis of M7 because many other types of AML may express one of these markers.111 Because of the common presence of fibrosis in this type of leukemia, immunohistochemical markers may be necessary for the diagnosis. CD61, von Willebrand factor, and Ulex europaeus are commonly positive in the immature cells by this method. The blasts are also commonly CD31+ but this marker is not sufficiently lineage specific to use alone. The disease seems to differ significantly in adults and children.

Pediatric M7 either occurs in the first 6 months of life and is most often associated with the t(1;22)(p13;q13), involving the RBMI5 and MKL1 genes,112 or it occurs in children 2 years of age or older with Down’s syndrome and trisomy 21.113,114 Adult M7 is almost always associated with multilineage dysplasia, frequently has cytogenetic abnor-malities similar to those seen in myelodysplasia,115 and is probably best classified as AML with myelodysplasia-related changes rather than as acute megakaryoblastic leukemia. Morphologic, immunophenotypic, and molecular genetic overlap may exist between M6 and M7, especially in adults, a finding suggesting that these diseases are closely related.116,117

Transient myeloproliferative disorder associated with Down’s syndrome is a proliferation that occurs in neonates who have the overall features of MDS or AML, particularly acute megakaryoblastic leukemia.118-120 These proliferations do not show t(1;22), although they show +21 and may have other karyotype abnormalities, and they usually regress in 1 to 3 months. It is not possible to predict, by morphologic features, which cases are transient. Even with regression in the neonatal period, some patients with Down’s syndrome will develop true acute megakaryoblastic leukemia, usually between 1 and 4 years of age.

acute myeloid leukemia with recurrent cytogenetic abnormalities

A major advance of the WHO classification of acute leuke-mias is the creation of a new category of AMLs that have recurring cytogenetic abnormalities. Recurring cytogenetic abnormalities in acute leukemia are among the most impor-tant prognostic indicators.95,96,121 Currently, this category in the WHO classification includes only the most common

cytogenetic and molecular genetic abnormalities. A major difference in this disease category of the WHO is that some cases with recurring cytogenetic abnormalities are consid-ered acute leukemia even if the bone marrow blast cell count is lower than 20%.

Acute Myeloid Leukemia with t(8;21)(q22;q22) (RUNX1/RUNX1T1)AML with t(8;21) has fairly unique morphologic, immuno-phenotypic, and clinical features and is a separate category in the WHO classification. These leukemias represented a subgroup of M2 AMLs in the FAB classification. This trans-location involves the ETO (or RUNX1T1) gene of chromo-some 8 and the AML1 (or RUNX1) gene of chromosome 21 that encodes a component of the core binding factor involved in normal hematopoiesis.122 These leukemias have a gener-ally better prognosis than do other AMLs and demonstrate characteristic M2 morphologic features (Fig. 43-9).123 The blast cells have large salmon-colored granules. The blast cells also have perinuclear clearing or hofs and frequently contain Auer rods. These cases also have characteristic immunophenotypic features. Most cases of t(8;21) AML aberrantly express the B-lineage associated markers CD19 and PAX5 on the blast cells that are also characteristically CD34+.124,125 The combination of morphologic features with CD19 and CD34 expression is fairly specific for this cyto-genetic translocation,90 but CD19 expression alone in AML is not sufficient for this diagnosis.111 Up to half of t(8;21) leukemias also express the CD56 antigen, a marker more commonly seen on natural killer (NK) cell lymphocytes.126 CD56 expression in t(8;21) leukemia is reported to be asso-ciated with a shortened time of remission and decreased survival. Rare cases of t(8;21) AML test negative for the commonly used myeloid-associated antigens CD13 and CD33, but they test positive for myeloperoxidase on cyto-chemical analysis.127 The RUNX1/RUNX1T1 fusion is easily detected by reverse transcriptase PCR (RT-PCR),128 but patients may remain RT-PCR positive even during remis-sion. This finding limits the use of routine RT-PCR testing for residual disease in these patients.129

Figure 43-9 n Acute myeloid leukemia (M2) with t(8;21). The blasts have numerous cytoplasmic granules, including large, chunky, salmon-colored granules that are characteristic of leukemias with this cytogenetic abnormality.C

BoNe maRRow n 1549

Acute Myeloid Leukemia with inv(16)(p13q22) or t(16;16)(p13;q22) (CBFB/MYH11)AML with inv(16) correlates with acute myelomonocytic leu-kemia with abnormal eosinophils (M4Eo) of the FAB classifi-cation and demonstrates an increase in eosinophils that have abnormal, large basophilic granules (Fig. 43-10).130 These eosinophils test weakly positive for chloracetate esterase, in contrast to normal eosinophils in other leukemias, but this cytochemical study is usually not neces-sary for the diagnosis. The significance of the finding of abnormal eosinophils is that it is associated with the presence of inv16(p13q22) or other abnormalities that involve this region of chromosome 16, including t(16;16)(p13;q22).130-132 These abnormalities result in fusion of the core binding factor beta (CBFB) gene to the smooth muscle myosin heavy-chain (SMMHC or MYH11) gene, both on chromosome 16. Disruptions of the core binding factor,

an important element of normal hematopoiesis, are also caused by the RUNX1/RUNXT1 fusion of t(8;21) leukemias and both leukemia types have improved prognosis when compared with other AMLs. Some cases of AML have abnor-mal eosinophils but do not fulfill the FAB cytochemical criteria for M4. Despite this situation, the cases have similar clinical outcomes and the same cytogenetic abnormalities. These cases are sometimes referred to as M2EO. Approxi-mately one third of AMLs with this cytogenetic abnormality will not have abnormal eosinophils and will be detected only by karyotype or molecular genetic studies. The abnor-mal eosinophils are particularly difficult to identify on peripheral blood specimens. Immunophenotyping studies show myeloid antigen expression, often with a subset of cells expressing monocyte-associated markers such as CD14 or CD64. A subset of cases has aberrant expression of CD2. Inversion of chromosome 16 may be difficult to identify by karyotype analysis, and FISH or RT-PCR studies in suspi-cious cases should be performed.

Acute Promyelocytic Leukemia (Acute Myeloid Leukemia with t(15;17)(q22.2;q12)(PML/RARA))Acute promyelocytic leukemia, or FAB M3, is another example of a disease with morphologic features that correlate with karyotypic and prognostic features (Fig. 43-11).133 These patients have a high occurrence of disseminated intravas-cular coagulopathy and generally respond favorably to therapeutic regimens that include all-trans-retinoic acid.134 In acute promyelocytic leukemia, abnormal promyelocytes predominate and are counted as blast cells. Different mor-phologic variants have been described,135,136 and the common denominator of the variants is the presence of bilobed nuclei that have been likened to butterfly wings. In the most common type, the cells demonstrate abundant cytoplasmic granules; cells with numerous Auer rods, termed faggot cells, may be identified. Some cases, however, do not have obvious cytoplasmic granules, may have basophilic cyto-plasm, and have folded bilobed nuclei that may be mistaken for monocytes. These cases are referred to as the micro-granular variant of acute promyelocytic leukemia, or M3v. Both types are strongly myeloperoxidase positive in virtu-

Figure 43-10 n Acute myelomonocytic leukemia with abnormal eosino-phils and inversion 16 (M4Eo). Numerous monocytoid blasts are present as well as abnormal eosinophils with abnormal, basophilic granules that are characteristic of this disorder.

Figure 43-11 n Acute promyelocytic leukemia (M3). A, The aspirate smear contains cells with folded, “monocytoid” nuclei and fine cytoplasmic granules. B, The biopsy specimen has sheets of cells with abundant pink cytoplasm.

A B

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ally all cells by cytochemical analysis or by flow cytometric study. A subpopulation of cells may also test positive for nonspecific esterase. Immunophenotyping studies confirm the myeloid lineage of the cells and usually demonstrate the blast cells to test negative for human leukocyte antigen (HLA)-DR, an antigen that is usually expressed in the other types of AML.111,137 The blasts may also aberrantly express the T-cell–associated antigen CD2, usually without CD7 expression. Apart from the morphologic features of typical acute promyelocytic leukemia, no one feature is sufficient for the diagnosis. A combination of findings of strong myeloperoxidase positivity with loss of HLA-DR, however, can be helpful in identifying cases of the microgranular variant. Most cases of acute promyelocytic leukemia, includ-ing the microgranular variant, have the cytogenetic abnor-mality t(15;17)(q22;p21), which fuses the retinoic acid receptor-alpha (RARA) of chromosome 17 to the promyelo-cytic leukemia gene (PML) of chromosome 15. Cytogenetic detection of t(15;17) by routine karyotyping or molecular methods is essential to define the disease further, because variant translocations may occur in cases of acute promy-elocytic leukemia. The best described variant translocations involve the RARA gene on chromosome 17 with the ZBTBI6 (PLZF) gene at 11q23, the NUMA1 gene at 11q13, the NPM1 gene at 5q35, or the STAT5b gene at 17q11. Although most of these variant translocations are responsive to all-trans-retinoic acid, translocations involving ZBTB16 or STAT5b are not responsive and require different treat-ment.138,139 Monitoring of t(15;17) can be easily performed by RT-PCR analysis,140 and the presence of this abnormality by RT-PCR after treatment correlates with an increased risk of relapse.141,142

Acute Myeloid Leukemia with 11q23 (MLL) AbnormalitiesSome AMLs are associated with various cytogenetic trans-locations or cryptic abnormalities involving the MLL gene of chromosome band 11q23,143 and those with a t(9;11) are now a separate category of the WHO classification.144 These cases usually meet criteria for FAB M4 or M5, but no dis-tinctive features suggest an MLL rearrangement in a given case of AML. The blasts often express monocytic markers such as CD14 or CD64 and often express CD56. The t(9;11)(p22;q23), involving the MLLT3 gene on chromo-some 9, is most commonly associated with monoblastic morphologic features, but various 11q23 abnormalities may be associated with other types of AML and even ALL. Abnormalities involving the MLL gene generally confer a poor prognosis, but patients with t(9;11) appear to fare better than do those with other MLL translocations.145 Administration of topoisomerase II inhibitors may also induce MLL breakpoints146 and result in therapy-related AMLs with abnormalities of 11q23 and monoblastic fea-tures. However, therapy-related AML with MLL abnormali-ties is not included in this category and is a separate category of therapy-related AML/MDS. Rearrangements of the MLL gene may not be evident by routine karyotype analysis in all cases. Various FISH, multiplex PCR, and Southern blot methods are performed in some laboratories, but are less available than are tests for other translocations. PCR methods miss some translocations and may detect evidence of an MLL abnormality in healthy patients when very sensi-tive methods are employed.147 Therefore, the use of PCR analysis for the monitoring of residual disease in these

patients is controversial148 and quantitative methods are probably the best approach.

Acute Myeloid Leukemia with Myelodysplasia-Related ChangesThe WHO classification includes cases of AML with associ-ated dysplasia, a history of MDS or myelodysplasia-related cytogenetics as a separate disease group. This category of AML usually occurs in older adults and may arise from a preexisting MDS or may present de novo. Both types have a generally poor prognosis.45 AML with multilineage dys-plasia is defined by the WHO as having 20% or more bone marrow blasts with dysplastic changes in at least 50% of the cells of at least two of the non–blast cell lines. The criterion of 50% of cells is somewhat arbitrary but it is important that dysplasia is not assigned to a lineage based on a single cell. Dysplastic changes of this category of AML are identical to those of myelodysplasia. Unfortunately, cases of AML with a high blast cell percentage in the bone marrow may not have sufficient non–blast cell elements to meet the diagnos-tic criteria of AML with multilineage dysplasia, but such cases may now be placed in the category based on cytogen-tic studies or a history of MDS. The immunophenotypic features of the blast cells are nonspecific and resemble those of other types of AML. Results of cytogenetic studies usually show abnormalities similar to those in myelodysplasia, including abnormalities of chromosomes 5 and 7 and complex abnormalities.

Therapy-Related Acute Myeloid LeukemiaTherapy-related AML is similar to therapy-related myelo-dysplasia (see earlier), except the blast cell count is 20% or greater in the bone marrow. Both therapy-related myelodys-plasia and AML have a poor prognosis and share morpho-logic, immunophenotypic and cytogenetic features. As mentioned earlier, these disorders are usually related to alkylating agents (usually with features essentially identical to AML with multilineage dysplasia) and to topoisomerase II inhibitors (often with features of AML with MLL abnor-malities). However, therapy-related AMLs may also occur after radiation therapy and often occur in patients who have received a combination of therapies. Prior therapy may also result in AMLs with the recurrent cytogenetic abnor-malities described earlier. Some of these cases have mor-phologic features similar to the features of those types of AML, but they may also show multilineage dysplasia that is not usually present in the AMLs with recurrent cytoge-netic abnormalities.149,150

other acute myeloid leukemia types

Some less common AML types of myeloid neoplasms have distinctive morphologic or molecular genetic features.

AML with maturation and increased basophils (M2-baso) has been described as a specific type of AML associated with abnormalities of the short arm of chromosome 12 or t(6;9)(p23;q34). Basophils may also be associated with t(9;22)-positive AMLs, including myeloid blast crisis of CMLs. Some of these cases may fall under the category of acute basophilic leukemia of the AML, not otherwise cate-gorized group in the WHO, but they appear to be a heter-ogeneous group of diseases. The presence of basophils in t(6;9) AML may also be seen with other FAB types of AML, including M1 and M4.151,152 In addition to basophilia, t(6;9)-

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positive AMLs are associated with relative erythroid hyperplasia and dysplastic bone marrow changes and are now a distinct category in the WHO classification. The t(6;9)(p23;q34) results in a fusion of the DEK gene on chromosome 6 with the CAN/NUP214 gene on chromosome 9. This type of AML also appears to be frequently associated with FLT3 mutations. This type of leukemia has a generally poor prognosis, but some patients appear to do well with aggressive therapy that includes hematopoietic stem cell transplantation. Although the presence of an increase in basophils in AML may suggest any one of these cytogenetic abnormalities, no morphologic subtype of disease is specific for any one of these cytogenetic abnormalities and a diag-nosis of M2-baso or acute basophilic leukemia is not of significance without appropriate cytogenetic studies. There-fore, I consider the presence of basophils in AML as a clue to possible abnormalities to be further investigated, but not as a marker of a specific AML type.

Myelodysplasia and AML with t(3;5)(q25;q35) appear to represent a distinct subset of myelodysplasia-associated dis-eases.153,154 Unlike most cases of AML with multilineage dysplasia and myelodysplasia, this disease tends to occur in young adults and has a male predominance. This disorder may occur with a wide range of blast cell counts, thus ful-filling criteria for refractory cytopenia with multilineage dysplasia and less than 5% blast cells to overt acute leuke-mia at presentation. In addition to the presence of multilin-eage dysplasia, erythroid hyperplasia often occurs and may meet criteria for acute erythroid leukemia (see earlier). Auer rods are frequently seen, even in the cases with lower blast cell counts. The morphologic and immunophenotypic features of these cases are otherwise nonspecific. The t(3;5) results in fusion of the nucleophosmin (NPM1) gene and the myeloid leukemia factor 1(MLF1) gene. Although early studies of this leukemia type suggested a poor prognosis, more recent studies suggested a more favorable prognosis with hematopoietic stem cell transplantation.154

Myelodysplasia and AML with multilineage dysplasia associated with an inversion of chromosome 3 at q21q26 or translocations involving this chromosome region are dis-tinct from the t(3;5) AMLs discussed earlier.155,156 The inv(3) AML most commonly involves the ecotropic virus insertion site 1 (EVI1) gene, which may also be disrupted with a t(3;3)(q21;q26) or with other translocations. This disease is associated with multilineage dysplasia and thrombocyto-sis with giant platelets and is now a distinct category in the WHO classification. The most distinctive bone marrow feature is the presence of small, unilobated, or bilobated megakaryocytes. The megakaryocytes are similar to those seen in the 5q− syndrome of myelodysplasia, but they are accompanied by the presence of multilineage dysplasia and an increase in blast cells in inv(3) AML, features not seen with 5q− syndrome. This disease is usually associated with a poor prognosis.

Many other genetic abnormalities also occur in AML, but their significance is not yet completely understood. These include both balanced translocations and gene mutations. The significance of point mutations, as well as balanced translocations, involving the MLL gene is discussed earlier. Mutations of the FLT3 gene are among the most common genetic abnormalities in AML; they occur in 20% to 28% of adult AMLs and in 11.5% of all de novo pediatric AMLs.157-160 FLT3 (also known as STK1 and flk2) is a tyro-

sine kinase gene that is currently the subject of much study. FLT3 mutations occur in almost any type of AML, but they appear to be most common in acute promyelocytic leuke-mia, in AMLs with a normal karyotype, and in the rare cases of t(6;9) AML. FLT3-positive AML in children is associated with older age (median age, 13.4 years) and in many studies is associated with a worse outcome than is FLT3-negative AML. These mutations are associated with decreased disease-free survival in adult AML. Because of the relatively high frequency and clinical significance of these mutations in AML, FLT3 mutation testing has become common in many institutions.

Mutations of the CEBPA gene occur in 7% to 11% of cases of AML.161,162 This gene encodes the basic zipper (B-ZIP) transcription factor CCAAT/enhancer binding protein-alpha (C/EBPα), which is critical for normal differentiation of mature granulocytes. AMLs with CEPBA mutations are associated with intermediate-risk cytogenetics and are more commonly associated with FAB M1 or M2 morphologic features (although virtually all FAB types are reported). Detection of a CEPBA mutation is generally associated with a favorable prognosis, although patients with both FLT3 and CEPBA mutations have an intermediate prognosis.163

Mutations of the NPM1 gene, which is distinct from the MLF1/NPM1 fusion of t(3;5) described earlier, have been reported as the most common molecular genetic abnormal-ity in AML.164-168 Mutation of this gene occurred in 27.5% of cases in one study and in almost half of patients with a normal karyotype. NPM1 mutations in AML appear to be more common in de novo AML and are unusual in acute promyelocytic leukemia and t(6;9) AML. AML with this mutation is more common in female patients and is associ-ated with high white blood cell counts and platelet counts, as well as high bone marrow blast cell counts. FLT3 muta-tions are also common in these patients; however, patients with NPM1 mutations that are not associated with FLT3 mutations have an improved response to therapy, whereas patients with wild-type NPM1 and mutated FLT3 have a worse prognosis.

Acute Lymphoblastic Leukemia

ALL is the most common malignant disease in children, with relatively high cure rates, but it also affects adults, with a more dismal prognosis.169 ALL is divided into three mor-phologic groups (L1, L2, L3) in the FAB classification, without consideration of the immunophenotype of the blast cell population (Figs. 43-12 to 43-14). In children, L2 mor-phologic features may be associated with a worse prognosis, but this finding may be related to other immunophenotypic and cytogenetic factors and is not necessarily an indepen-dent prognostic factor.170,171 Blasts with abundant cytoplasm, irregular nuclear membranes, or prominent nucleoli should represent 25% or more of total blast cells before a diagnosis of L2 is made.172 The WHO classification separates the lym-phoblastic leukemias by immunophenotype and recognizes the great overlap between these diseases and lymphoblastic lymphomas. Cases are generally considered leukemia if more than 25% bone marrow involvement is present and lymphoma if the marrow contains 25% or fewer lymphoblasts.

Precursor B-cell ALLs are proliferations of immature B cells and represent the majority of all ALLs. The blasts have

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fine nuclear chromatin, small indistinct nucleoli, and scant, slightly basophilic cytoplasm. The blast cells are often homogeneous in size and shape, but some cases may show marked variation in nuclear size and have abundant cyto-plasm (L2 morphology). Subclassification into immuno-logic subtypes usually defines cases as pro-B if CD10 negative, common-B if CD10+ but negative for cytoplasmic µ, and pre-B if positive for both cytoplasmic µ and CD10.173 When cytogenetic findings are taken into account, these immunophenotypic subtypes add little additional informa-tion. For this reason, I prefer the general term of precursor B-cell ALL, which covers all subtypes. Morphologic features are not helpful in the prediction of certain cytogenetic abnormalities and cytogenetic studies are essential to iden-tify prognostically significant disease groups.174 However, some immunophenotypic features may be clues to recurring cytogenetic abnormalities in this disease.175

Precursor T-cell ALL represents 15% to 20% of all ALL cases and is immunophenotypically identical to most cases

of lymphoblastic lymphoma.176,177 Patients frequently present with a mediastinal mass consisting of T lympho-blasts. In most cases, the blast cells are morphologically indistinguishable from those of precursor B-cell tumors. Subclassification of T-cell ALL by immunophenotype is also described173 but is not necessary. Rare cases of T-cell ALL have basophilic cytoplasm and cytoplasmic vacuoles that may erroneously suggest L3 leukemia if immunophenotyp-ing to assign lineage is not performed.

Burkitt’s leukemia/lymphoma, also known as mature B-cell ALL, includes most cases of FAB L3 ALL, although some precursor lymphoid neoplasms may demonstrate the characteristic cytoplasmic vacuoles of L3 leukemia. Burkitt’s leukemia is a mature B-cell proliferation that demonstrates large leukemic cells with clumped nuclear chromatin with multiple nucleoli. The cells have moderately abundant basophilic cytoplasm with or without cytoplasmic vacuoles. The vacuoles contain lipid that stains positive for oil red O, but these vacuoles are not required for a diagnosis of Burkitt’s leukemia. Because of similarities in morphologic features, immunophenotype, and karyotype between these leukemias and Burkitt’s lymphoma,178 they are combined as different presentations of a single disease in the WHO classification.

Both cytogenetic and immunophenotyping studies are essential in the workup of ALL. Immunophenotyping excludes minimally differentiated AML (M0), which has similar morphologic features and allows for the proper clas-sification of the ALL into the three immunologic types of precursor B-cell, T-cell, and Burkitt’s leukemia.179,180 Precur-sor B-cell ALLs almost always test positive for CD19, CD22, CD79a, and TdT and usually test positive for CD45 and CD10. The cells test negative for CD20 in more than half of cases and surface immunoglobulin expression is gener-ally not detectable. Loss or weak expression of CD45 is associated with an improved prognosis.181,182 Loss of CD10 is generally a poor prognostic indicator in precursor B-cell ALL,183,184 and loss of CD10 with aberrant CD15 expression is seen commonly in ALL of infants and therapy-related ALL of adults and is usually associated with 11q23 abnor-

Figure 43-12 n Acute lymphoblastic leukemia (L1). The blast cells are uniform in size with scant cytoplasm. This case represents T-cell acute lymphoblastic leukemia, but the morphologic features are not helpful in predicting the immunophenotype.

Figure 43-13 n Acute lymphoblastic leukemia (L2). The blast cells are vari-able in size with multiple nucleoli and moderately abundant cytoplasm. Some contain vacuoles, but the number of vacuoles is not sufficient for a diagnosis of L3. This leukemia was of a precursor B-cell type in an infant with t(4;11). The cells lacked expression of CD10 and tested positive for CD15. This immunophenotype and cytogenetic abnormality are common in infant leukemias.

Figure 43-14 n Burkitt’s leukemia/lymphoma (L3). The blasts have mul-tiple nucleoli, basophilic cytoplasm, and abundant cytoplasmic vacuoles. The cells demonstrated a mature B-cell immunophenotype with surface immunoglobulin expression and no terminal deoxynucleotidyl transferase (TdT)–positive expression. Cytoplasmic vacuoles are not required for this diagnosis.

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malities in both patient groups. Rare cases of precursor B-cell ALL express both TdT and surface immunoglobulin light chains.185 These cases should not be confused with Burkitt’s leukemias, which are TdT negative. Precursor T-cell ALL does not always express surface CD3, but cytoplas-mic CD3 is usually detectable and other T-cell antigens, such as CD2, CD5, and CD7, are usually expressed, as well as TdT. Double expression of CD4 and CD8 may be present. CD10 expression is present in some cases of T-cell ALL and is associated with a good prognosis.179 Burkitt’s leukemia is a mature, monoclonal B-cell proliferation that expresses surface immunoglobulin light and heavy chains as well as CD20 and CD10, but not TdT.

Many cases of precursor B-cell and T-cell ALL express at least one myeloid-associated antigen and some express both CD13 and CD33.180,186 Although such aberrant antigen expression is useful in following the patient for residual disease, it should not be taken as an indication of bipheno-typic acute leukemia (see later). When myeloid antigens are expressed in ALL, they are usually present only on a subset of the blast cells. Virtually all the blast cells express antigens more typical of ALL and the detection of a subset of cells with myeloid antigen expression does not appear to have clinical significance.186

Cytogenetic and molecular genetic studies are used to identify prognostically significant disease groups in ALL.174,175 These recurrent cytogenetic subgroups are now specific disease groups in the WHO classification, because their prognostic significance is similar to that of recurrent cytogenetic abnormalities in AML. The Philadelphia chro-mosome is present in approximately 20% of adult and in fewer than 5% of pediatric ALL cases and confers a worse prognosis. This translocation, t(9;22)(q34;q11), produces a BCR/ABL1 fusion transcript. The p190 protein product is more commonly formed in ALL, in contrast to the p210 product seen in most patients with Philadelphia chromo-some–positive CML. Philadelphia chromosome–positive ALL often has aberrant expression of myeloid-associated antigens,180,187 and a high correlation exists with expression of CD13, CD33, and CD38 in ALL and the Philadelphia chromosome.188 Translocations involving chromosome band 11q23 are of prognostic significance that differs with the different translocations, but the most common 11q23 translocation in ALL, t(4;11)(q21;q23), confers a poor prognosis. Most of these translocations involve the MLL gene, and as mentioned these cases have a higher frequency of loss of CD10 and aberrant expression of CD15 than other ALLs.189 Some cytogenetic abnormalities that have prognos-tic significance are not detectable by standard karyotyping. Some 11q23 abnormalities involve deletions that cannot be readily visualized and t(12;21) is not usually detectable by karyotyping. The t(12;21)(p13;q22) is the most common cytogenetic abnormality in childhood ALL, present in at least 20% of cases, and identifies a group of patients with an improved prognosis.190 The blasts of this type of ALL characteristically show bright CD10 expression as well as aberrant myeloid antigen expression. Molecular genetic studies are necessary for identifying these abnormalities, which are difficult to discern. However, as mentioned earlier, some abnormalities of the MLL gene are detectable in a small number of cells in some healthy patients, and the use of PCR alone for the detection of MLL rearrangements

remains controversial.147,148 The t(1;19)(q23;p13.3) occurs in both children and adults and usually expresses both CD10 and cytoplasmic µ. This abnormality results from a fusion of the PBX and E2A (TCF3) genes. Other transloca-tions involving PBX may also occur. In the past, pediatric patients with ALL with PBX translocations had a worse prognosis, but this may no longer be true with current therapeutic approaches. In children, DNA ploidy analysis also provides information related to prognosis.191,192 Patients with hyperdiploid ALLs (>50 chromosomes or a DNA index ≥1.16) have improved survival over patients with other ploidy groups. Immunoglobulin and T-cell receptor gene rearrangements commonly both occur in a single case of leukemia and do not necessarily correlate well with the immunophenotype.193 The identification of these rearrange-ments, however, allows for the identification of patient-specific clones that can be monitored for residual disease by PCR analysis.194

Acute Leukemia of Ambiguous Lineage

Even with extensive immunophenotyping, the lineage of an acute leukemia may remain elusive. Acute undifferentiated leukemia is uncommon and shows undifferentiated blast cells that often express markers such as CD34, HLA-DR, TdT, and CD7 but do not consistently express lineage spe-cific markers. Bilineal acute leukemia and acute bipheno-typic leukemia were combined in the past as mixed lineage leukemia but are now better defined. Bilineal acute leukemia is uncommon and shows two morphologically and immu-nophenotypically distinct blast cell populations. The popu-lations are usually myeloid and precursor B or T lineage, but mixed precursor T and B lineage may rarely occur. Biphenotypic acute leukemia shows a single blast cell popula-tion, but the blasts express antigens of more than one lineage. The number of antigens expressed to constitute a biphenotypic immunophenotype is controversial, and some early studies required expression only of any two myeloid-associated and two-lymphoid associated antigens, no matter how nonspecific. This approach, however, would place cases of distinct ALL and AML subtypes, such as Philadelphia chromosome–positive ALL and t(8;21) posi-tive AML, into this category inappropriately and more spe-cific immunophenotypic criteria are required for a diagnosis of biphenotypic acute leukemia. The European Group for the Immunologic Classification of Leukemias proposed a scoring system for biphenotypic leukemias that weights antigen expression according to its specificity.173 My modification of this scoring system is presented in Table 43-12.195 More than two points (≥2.5) must be present for two different lineages before a case is designated as biphenotypic. The use of such a system limits the number of cases that are called biphenotypic and increases the number of cases of ALL with myeloid antigen expression and AML with lymphoid antigen expression. It is not clear whether this system truly defines a specific disease entity because cytogenetic analysis of these cases often shows evidence of MLL abnormalities or t(9;22).196 This latter group may be better characterized as Philadelphia chro-mosome–positive ALL with aberrant myeloid antigen expression. C


Myeloproliferative Neoplasms

The myeloproliferative neoplasms are clonal stem cell pro-liferations that demonstrate bone marrow hypercellularity with varying degrees of marrow fibrosis.197 They show great morphologic overlap but generally differ by which cell line is most proliferative. Although detailed descriptions of the morphologic features of the various myeloproliferative neo-plasms are useful in suggesting a specific disease,198 precise classification requires correlation of morphologic features with clinical, hematologic, and cytogenetic findings. A defi-nite diagnosis cannot usually be made by morphologic examination alone.

Chronic Myelogenous Leukemia, BCR-ABL1+

CML,199,200 the most common of the myeloproliferative neo-plasms, can occur at any age. It is defined by the presence of the Philadelphia chromosome or molecular genetic evi-dence of the BCR/ABL1 fusion product. In contrast to the t(9;22) of acute leukemias, which usually demonstrate the p190 BCR/ABL1 fusion protein, CML usually demonstrates the p210 fusion protein of this translocation,201 but a rare neutrophilic variant demonstrates a p230 protein. CML is primarily a proliferation of granulocytic cells, although multiple cell lines carry the Philadelphia chromosome. The peripheral blood findings are those of an elevated white blood cell count with granulocytes at all stages of matura-tion (Fig. 43-15). Segmented neutrophils may show abnor-mal nuclear segmentation. Myelocytes and metamyelocytes are present in high numbers in the peripheral blood and basophils are also increased in most cases. Abnormal baso-phils with “washed-out” granules may be identified. Platelet counts are usually elevated. The bone marrow is markedly hypercellular with a cellularity approaching 100% in most untreated cases (Fig. 43-16A). The myeloid-to-erythroid ratio of the bone marrow is markedly elevated, often greater than 10 : 1. Megakaryocytes are increased in number and

clusters of small megakaryocytes with hypolobated nuclei are usually present. Some degree of reticulin fibrosis is usually demonstrated in these cases, and marked increases in reticulin and collagen marrow fibrosis are associated with decreased survival.202 Pseudo-Gaucher histiocytes with abundant fibrillar birefringent cytoplasm are common in CML, reportedly present in 37% to 70% of cases,203 and this finding has been associated with improved survival.202 Leu-kemoid reactions in patients with infectious or other reac-tive conditions may have features similar to those of CML. These reactive proliferations do not usually show the baso-philia, degree of marrow cellularity, or megakaryocyte clus-tering characteristic of CML. However, cytogenetic or molecular genetic studies are indicated if the differential diagnosis includes CML.

CML usually manifests in chronic phase, which is essen-tially defined by the lack of features of accelerated or blastic phase. Many patients with chronic phase of CML eventually undergo transformation to a more aggressive phase of disease. Any one of a variety of parameters defines acceler-ated phase of CML, but the criteria for the aggressive phases of CML are inconsistent in the literature.204,205 Most criteria require the presence of either 10% or 15% or more blasts in the peripheral blood or bone marrow or the presence of 20% or more blast cells in addition to promyelocytes in the blood or marrow for a diagnosis of accelerated phase. Other cri-teria for accelerated phase include the development of myelofibrosis, acquisition of additional chromosomal abnormalities (clonal evolution), elevations of basophils and eosinophils to 20% or more of blood cells, or any of the following that do not respond to conventional therapies: persistence of anemia or thrombocytopenia, marked white blood cell elevations, platelet elevations of 1,000,000/µL or more, or increasing splenomegaly. The WHO criteria for accelerated phase of CML are given in Table 43-13. Blastic phase, or blast crisis, of CML is now defined by WHO as the presence of 20% or more blast cells in the peripheral blood or bone marrow, the development of an extramedul-lary myeloid tumor, or the presence of large aggregates and clusters of blasts in a bone marrow biopsy specimen.

Detection of the t(9;22)(q34;q11) by karyotype analysis, FISH, or RT-PCR is essential for the diagnosis of CML.

taBle 43-12Modified Scoring System for Biphenotypic Acute Leukemia*

Points B-Cell Lineage T-Cell Lineage Myeloid Lineage

2 Cd79a c/sCd3 myeloperoxidase cytochemistry

cigm anti-tCRα/βcCd22 anti-tCRγ/δ

1 Cd19 Cd2 Cd13Cd10 Cd5 Cd33Cd20 Cd8 Cd65

Cd10 anti-mpoCd117

0.5 tdt tdt Cd11cCd24 Cd7 Cd14

Cd1a Cd15

*more than two points each for the myeloid and one of the lymphoid lineages are required for a diagnosis of biphenotypic acute leukemia.

Figure 43-15 n Chronic myelogenous leukemia. The peripheral blood white blood cell count is markedly elevated with increased numbers of granulocytes, including many myelocytes.

data from Bene mC, Castoldi G, Knapp w, et al: proposal for the immunologic classification of acute leukemias. leukemia 9:1783-1786, 1995; and arber da, Snyder dS, Fine m, et al: myeloperoxidase immunoreactivity in adult acute lymphoblastic leukemia. am J Clin pathol 116:25-33, 2001.

igm, immunoglobulin m; mpo, myeloperoxidase; tCR, t-cell receptor; tdt, terminal deoxynucleotidyl transferase.

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Detection of this abnormality confirms the clonal or neo-plastic nature of the proliferation and excludes a leukemoid reaction that may mimic CML as well as helping to exclude other myeloproliferative neoplasms. Immunophenotyping studies are of little value in the chronic phase of CML but are helpful in defining the blast cell population of acceler-ated and blast phases of this disease.206,207 Although myeloid blast crisis of CML is most common, approximately one third of cases are lymphoid blast crises. Most lymphoid blast

crisis cases are of precursor B-cell lineage, but rare T-cell blast crisis cases occur. Lymphoid blast crisis is reported to have a better prognosis than myeloid blast crisis, and lym-phoid blast crisis has traditionally been defined as a blast cell proliferation that is TdT positive. More detailed immu-nophenotyping of these cases usually demonstrates expres-sion of other precursor B-cell markers, such as CD19 and CD10, but expression of myeloid-associated antigens, such as CD13 and CD33, is also common. Patients with a lym-phoid immunophenotype, irrespective of the expression of myeloid antigens, have improved survival.207 These cases are probably best classified as lymphoid blast crisis.

Bone marrow specimens of CML patients treated with interferon-alpha208 may be morphologically normal with normocellularity and a normal myeloid-to-erythroid ratio. The normal morphologic features do not necessarily indi-cate a complete cytogenetic response and karyotype analy-sis is still indicated. Treatment with imatinib mesylate (Gleevec) has become the primary treatment of CML. This tyrosine kinase inhibitor often results in morphologic remis-sion with normalization of the marrow cellularity and even erythroid hyperplasia. Cytogenetic and molecular genetic studies are usually indicated in these cases to identify resid-ual disease that is not morphologically apparent.

Figure 43-16 n Bone marrow biopsy specimens of the various myeloproliferative neoplasms in chronic phase. A, Chronic myelogenous leukemia is mark-edly hypercellular with a high myeloid-to-erythroid ratio and clusters of small megakaryocytes. B, The megakaryocytes of essential thrombocythemia are larger with more abundant cytoplasm. C, The marrow of primary myelofibrosis is often more fibrotic with atypical megakaryocytes that often have hyper-chromatic nuclei. Trabecular bone is often thickened in this disorder as well. D, The proliferative phase of polycythemia vera may resemble essential thrombocythemia with large megakaryocytes, but increased numbers of erythroid precursors are evident. The spent phase of polycythemia vera is usually indistinguishable from the marrow of primary myelofibrosis.

A B

C D

taBle 43-13World Health Organization Criteria for Accelerated Phase of Chronic Myelogenous Leukemia

any one or more of the following features:peripheral blood or bone marrow blasts of 10%-19%peripheral blood basophils of ≥20%persistent thrombocytopenia of <100 × 109/l unrelated to therapypersistent thrombocytosis of >1000 × 109/l unresponsive to therapyincreasing spleen size and increasing white blood cell count unresponsive

to therapyCytogenetic evidence of clonal evolution

adapted from Swerdlow SH, Campo e, Harris Nl, et al (eds): wHo Classification of tumours of Haematopoietic and lymphoid tissues. lyon, France, iaRC press, 2008. C


Neutrophilic CML209 appears to be a less aggressive variant of CML. This disease is associated with proliferation of more mature granulocytes, usually at the segmented neu-trophil stage of development. These patients have less severe clinical symptoms and are slower to progress to a blastic stage. These patients have a Philadelphia chromosome, but the BCR/ABL1 fusion protein is larger than that usually seen in CML, at 230-kD. This condition differs from chronic neutrophilic leukemia, which is a rare disease defined as having persistent neutrophilia with a white blood cell count of 25 × 109/L or greater, a hypercellular bone marrow without increased blasts, and hepatosplenomegaly. The diagnosis also requires the absence of infection, underlying tumor, t(9;22), or another myeloproliferative neoplasm, MDS, or mixed MDS/myeloproliferative neoplasm. The prognosis of chronic neutrophilic leukemia is variable; some cases transform to acute leukemia.210

Philadelphia chromosome–negative CML is a rare occur-rence and such a diagnosis should probably no longer be made. Cryptic BCR/ABL1 translocations may occur that cannot be identified by routine karyotype analysis. When this situation is suspected, molecular genetic studies, such as FISH or RT-PCR analysis, are indicated. If the results of these studies are negative, other diagnostic possibilities, such as CMML or atypical CML (aCML; described later), must be considered.211

Essential Thrombocythemia

ET is a bone marrow proliferation that primarily is charac-terized by an elevation in peripheral blood platelets. The platelet count is usually greater than 1000 × 109/L and must be more than 600 × 109/L using the Polycythemia Vera Study Group and 450 × 109/L or more in the more recent WHO criteria for ET (Table 43-14).42,212 Modified criteria for this disease have also been proposed, some of which allow for platelet counts between 400 and 600 × 109/L.213,214 Cases with features of ET probably represent two different diseases, one clonal and one reactive.215 Without clonality assays, which are challenging to perform, however, it may be difficult to differentiate the two groups. Patients with the nonclonal form of the disease may include those with abnormalities of the thrombopoietin gene, and these patients appear to be at a decreased risk of developing thrombo-sis.215,216 Both types show similar peripheral blood and bone marrow features. The thrombocytosis may be accompanied by abnormal, large platelets. Leukocytosis, when present, is

usually mild, without the prominent left shift or associated increase in basophils seen in CML. The bone marrow is usually slightly to moderately hypercellular with increased numbers of megakaryocytes occurring in clusters (see Fig. 43-16B). The megakaryocytes tend to be large with abun-dant cytoplasm and multilobated nuclei,217 and they are larger than those seen in reactive conditions and CML. The myeloid-to-erythroid ratio is nearly normal and marrow fibrosis is minimal. Thrombotic and hemorrhagic complica-tions are the most frequent with this disease,218 and trans-formation to acute leukemia occurs in up to 5% of patients.79,219 With time, marked marrow fibrosis may occur producing a “spent” phase similar to that seen in polycy-themia vera (PV), but this event is also relatively uncommon.

The value of ancillary studies in ET is similar to that in CML, except studies for somatic point mutations in the JAK2 gene, which are performed in all cases of suspected ET. The discovery of mutations of JAK2 in many myelopro-liferative neoplasms made this a common test in evaluating these entities. JAK2 mutations occur in 23% to 57% of cases of ET and the detection is useful in determining that the proliferation is clonal.220-223 The detection of clonal cytoge-netic abnormalities is useful in determining that the mor-phologic changes represent a neoplastic rather than a reactive process, but most cases do not demonstrate cyto-genetic abnormalities.

Primary Myelofibrosis

Primary myelofibrosis (PM), also known as chronic idiopathic myelofibrosis, myelofibrosis with myeloid metaplasia or agnogenic myeloid metaplasia, occurs in elderly patients with a slight male predominance and usually manifests with leukoerythroblastic peripheral blood, massive splenomeg-aly, and bone marrow fibrosis.42,224-226 The peripheral blood changes include the presence of large, teardrop-shaped red blood cells, a granulocyte left shift often including rare myeloblasts, and giant platelets that are larger than a red blood cell (Fig. 43-17). Basophilia may be present and bare megakaryocyte nuclei are often seen in the blood and bone

taBle 43-14Criteria for Essential Thrombocythemia

1. Sustained platelet count >450 × 109/l or higher2. atypical (enlarged) megakaryocytic hyperplasia of the bone marrow

without a significant increase of left shift in granulocytes or erythroid cells

3. No evidence of polycythemia vera, primary myelofibrosis, BCR-ABL1 chronic myelogenous leukemia, myelodysplasia, or other myeloid neoplasm.

4. demonstration of JAK2 mutation or other clonal marker, or in the absence of JAK2 mutation, no evidence of reactive thrombocytosis.

adapted from Swerdlow SH, Campo e, Harris Nl, et al (eds): wHo Classification of tumors of Haematopoietic and lymphoid tissues. lyon, France, iaRC press, 2008.

Figure 43-17 n The peripheral blood changes of primary myelofibrosis characteristically show a triad of marked red blood cell poikilocytosis with abundant teardrop-shaped cells, a granulocyte left shift with rare blast cells, and abnormal giant platelets.C

BoNe maRRow n 1557

marrow. Although spleen enlargement occurs with many myeloproliferative neoplasms. The splenomegaly of PM is much more extreme than usually seen and may cause severe discomfort and wasting syndromes. The bone marrow may be hypercellular, particularly early in the disease when marrow fibrosis is less prominent. The myeloid-to-erythroid ratio is slightly increased and megakaryocytes are increased (see Fig. 43-16C). Most patients show marked marrow fibrosis, which may include collagen fibrosis. Clusters of atypical megakaryocytes remain prominent in association with the fibrosis, and megakaryocyte clusters in sinuses may be evident. Sclerosis of bone trabeculae also occurs in many patients. Lymphoid aggregates, of predominantly T cells, occur commonly in association with PM.

The diagnostic criteria proposed by an Italian consensus conference are listed in Table 43-15. These criteria require the presence of diffuse marrow fibrosis and the absence of the Philadelphia chromosome, in addition to any other two optional criteria when splenomegaly is present or four optional criteria when splenomegaly is absent.227 The most frequent causes of death in these patients are as follows: bone marrow failure (22%), including anemia, infections, and hemorrhage; transformation to AML (15%); and portal hypertension related to massive splenomegaly (11%).228 The WHO classification42 includes both a prefibrotic and a fibrotic stage of the disease. The fibrotic stage is similar to that described earlier. The prefibrotic stage, reportedly occurring in 20% to 30% of patients at presentation, is characterized by hypercellular bone marrow with little or no marrow fibrosis, but fairly typical peripheral blood changes and atypical megakaryocytes in clusters. These clusters occur adjacent to sinuses and bony trabeculae and are enlarged with cloudlike or balloon-shaped nuclei that are not usually seen in the other myeloproliferative neo-plasms. Without recognition of these atypical megakaryo-cyte, cases of PM in the prefibrotic stage may be interpreted as an unclassified myeloproliferative neoplasm.

Ancillary studies are useful to exclude the Philadelphia chromosome of CML; approximately 35% of patients will demonstrate a cytogenetic abnormality, with deletions of

chromosomal arms 20q and 13q most common.228-230 Muta-tions of JAK2 occur in 35% to 57% of patients with PM and these studies may be useful to confirm the presence of a clonal disease.220-223 Other ancillary studies are of limited utility with the exception of immunophenotyping of the blast cells of the cases that undergo blastic transformation. The spent phase of PV may have similar morphologic fea-tures (see later).

Polycythemia Vera

PV is a clonal proliferation, usually occurring in elderly patients, with a slight male predominance that manifests mainly as an expansion of the red blood cell mass.231,232 Because of the morphologic overlap between this disease and other myeloproliferative and reactive conditions, detailed criteria are described for this disorder (Table 43-16).43,232-234 The spleen is usually enlarged and erythropoie-tin levels are usually decreased in this disease.235 Two morphologic phases of disease occur.236 The proliferative or erythrocytotic phase may be characterized by elevations of red blood cells, white blood cells, mostly left-shifted granu-locytes, and platelets. Slight elevations in the peripheral blood basophil count may be present, but basophil counts are not as elevated as is usually seen in CML. The platelet count may exceed 600,000/µL, which may cause confusion with ET. Some patients may have associated iron deficiency with microcytic red blood cells. The bone marrow is usually moderately hypercellular with all cell lines involved (see Fig. 43-16D). In contrast to some other myeloproliferative neoplasms, however, the erythroid series is increased. Clus-ters of atypical megakaryocytes are common, similar to

taBle 43-15Italian Criteria for Primary Myelofibrosis

required Criteria

1. diffuse bone marrow fibrosis2. absence of the philadelphia chromosome or BCR/ABL1 in peripheral blood

Optional Criteria*

1. Splenomegaly of any grade2. anisopoikilocytosis with teardrop-shaped red blood cells3. presence of circulating immature myeloid cells4. presence of circulating nucleated red blood cells5. presence of clusters of megakaryocytes and abnormal megakaryocytes in

bone marrow sections6. myeloid metaplasia

*three optional criteria are needed if one is splenomegaly, and four are required if splenomegaly is not present.

taBle 43-16World Health Organization Criteria for the Diagnosis of Polycythemia Vera

the diagnosis requires both major and one minor criteria or the first major and two minor criteria.

Major

1. Red cell mass >25% of normal predicted mean value or hemoglobin <18.5 g/dl (men) or <16.5 g/dl (women)

2. No cause of secondary erythrocytosisa. absence of familial erythrocytosisb. No elevation of erythropoietin resulting from

Hypoxia (arterial saturation <92%)High oxygen affinity hemoglobintruncated erythropoietin receptorerythropoietin producing tumors

3. Splenomegaly4. Clonal cytogenetic abnormality (other than philadelphia chromosome or

BCR/ABL gene rearrangement)5. endogenous erythroid colony formation in vitro

Minor

1. thrombocytosis/platelet count >400 × 109/l2. white blood cell count >12 × 109/l3. Bone marrow biopsy showing panmyelosis with prominent erythroid,

granulocytic, and megakaryocytic proliferation4. low serum erythropoietin levels5. endogenous erythroid colony formation in vitro

adapted from Swerdlow SH, Campo e, Harris Nl, et al (eds): wHo Classification of tumours of Haematopoietic and lymphoid tissues. lyon, France, iaRC press, 2008.

data from Barosi G, ambrosetti a, Finelli C, et al: the italian consensus conference on diagnostic criteria for myelofibrosis with myeloid metaplasia. Br J Haematol 104:730-737, 1999.

the world Health organization classification also includes a prefibrotic stage of primary myelofibrosis that does not meet these criteria and incorporates use of JAK2 mutation studies (see text and reference 42).

C


those seen in ET. Bone marrow fibrosis may be minimal in this stage of the disease. The spent phase of PV is associated with marked marrow fibrosis and shows peripheral blood and bone marrow changes similar to those seen in PM with leukoerythroblastic peripheral blood changes, splenomeg-aly, and marrow fibrosis. Differentiation between these two diseases may not be possible without a history of the earlier phase of PV; however, the megakaryocytes of PV usually have hyperchromatic nuclei compared with the cloudlike nuclei of megakaryocytes in PM. Because of the overlap in features between ET and PV, some cases may be resolved only by red blood cell mass studies. Red blood cell mass may be decreased in PV patients who also have iron defi-ciency, and the studies may have to be repeated after iron therapy if PV is suspected. Reactive or secondary polycy-themia must also be excluded and may be related to smoking, lung and renal disease, erythropoietin-producing tumors, congenital conditions causing erythropoietin overproduc-tion, and exogenous administration of erythropoietin. The atypical megakaryocytic hyperplasia of PV is not seen in these conditions. In approximately 10% of patients, PV will undergo transformation to AML within 15 years and up to half these patients will develop acute leukemia over 20 years.232 Most patients die of thrombosis or hemorrhage.

Again, the absence of a Philadelphia chromosome is essential for the diagnosis. Other karyotype abnormalities may be detected, with chromosome 20q deletions being the most common.237,238 These abnormalities are most common in the spent phase; however, they are not specific for PV.239 Various point mutations occur in association with polycy-themias, particularly in the congenital or familial forms.240 JAK2 mutations occur in 65% to 97% of cases of PV and this finding is extremely useful in excluding reactive polycythemia.220-223

Other Myeloproliferative Neoplasms

Other myeloproliferative neoplasms have been described and include chronic neutrophilic leukemia, chronic eosino-philic leukemia, hypereosinophilic syndrome, and unclas-sifiable cases. All cases should be studied for the Philadelphia chromosome, to exclude unusual presentations of CML. Cases that are Philadelphia chromosome negative with per-sistent neutrophilia without a reactive cause or evidence of another myeloproliferative neoplasm or MDS are consid-ered chronic neutrophilic leukemia.42 These patients must have a peripheral blood white count of 25 × 109/L or greater with more than 80% segmented neutrophils or bands and must also have hypercellular bone marrow and hepato-splenomegaly. Many of the other Philadelphia chromo-some–negative chronic myeloid proliferations fall into the category of aCML (described later).

Chronic eosinophilic leukemia and idiopathic hypereosino-philic syndrome241 are defined as an unexplained peripheral blood proliferation of more than 1500/µL of eosinophils for 6 months or more with bone marrow eosinophilia, associ-ated hypercellularity, and associated tissue damage. Tissue damage is usually cardiac, presumably related to release of eosinophil granule contents. Other causes of eosinophilia, including parasite infections, allergies, and elevations of eosinophils associated with other malignancies, including CML, must be excluded. Once it is determined that the proliferation is related to a myeloproliferative neoplasm, the

term idiopathic hypereosinophilic syndrome is probably not appropriate, and even cases without clonal cytogenetic abnormalities have demonstrated clonality by analysis of X-chromosome inactivation patterns.242 Numerous clonal cytogenetic abnormalities are reported in chronic eosino-philic leukemia, but cryptic fusions of FIP1L1 and PDGFRA, t(5;12)(q33;p13), involving PDGFRB and ETV6, and other abnormalities involving PDGFRB are most commonly asso-ciated with this disorder.243,244 Disorders with PDGFRA and PDGFRB abnormalities, as well as those with FGFR1 abnor-malities (see further on) are now considered discrete enti-ties in the 2008 WHO classification. The FIP1L1/PDGFRA fusion protein has tyrosine kinase activity that is inhibited by imatinib mesylate. However, this cryptic abnormality can be detected only by molecular genetic (FISH or PCR) studies. Myeloproliferative syndromes with translocations involving 8p11 may also be associated with eosinophilia.245 These disorders are frequently associated with non- Hodgkin’s lymphomas, including T-cell lymphoblastic lym-phoma, and appear to transform to acute leukemia more commonly than chronic eosinophilic leukemia. The fibro-blast growth factor receptor-1 (FGFR1) gene of chromo-some band 8q11 most commonly fuses with the ZNF198 gene of 13q12 for a t(8;13)(q11;q12), but other 8q11 trans-locations may occur.246

Other patients may present with bone marrow hyper-cellularity suggestive of a myeloproliferative neoplasm that does not fit well into any of the previously described catego-ries after correlation with cytogenetic and other laboratory studies. Many of these cases represent early presentations of a specific type of myeloproliferative neoplasm that will progress to have more typical features over time. Other cases may represent end-stage disease with bone marrow fibrosis in which the original disease cannot be determined. Rare cases of unclassified myeloproliferative neoplasm are associated with mast cell disease. Some cases may be best classified as CMML or aCML after more careful examina-tion,211 as described later.

Myelodysplastic/Myeloproliferative Neoplasms

Some proliferations have features of both myeloproliferative neoplasms and MDSs (MDS/MPN).247,248 They manifest with cytopenias and dysplastic changes of any cell line, similar to the MDSs, and elevated white blood cell counts, hyper-cellular bone marrow with fibrosis, and organomegaly, fea-tures more commonly associated with myeloproliferative neoplasms. The presence of fibrosis alone in a case that is otherwise typical of myelodysplasia is not sufficient to use this category. The three best-defined MDS/MPNs are aCML, CMML, and juvenile myelomonocytic leukemia. Features that are helpful in differentiating the chronic phase of CML from aCML and CMML in adults are listed in Table 43-17.

Atypical Chronic Myeloid Leukemia, BCR-ABL−

aCML249,250 is a Philadelphia chromosome–negative and BCR/ABL1-negative proliferative neoplasm that affects elderly patients with an apparent male predominance.

C

BoNe maRRow n 1559

Patients have some features of usual CML with spleno-megaly, an elevated white blood cell count of predominantly granulocytic cells, anemia, and normal or decreased platelet counts. However, patients with aCML are usually older and some may have an initial presentation more typical of myelodysplasia with a low white blood cell count with evolution into aCML.251 The white blood cells are left shifted with immature granulocytes, including blast cells, promy-elocytes, and myelocytes, representing 10% to 20% of cells and dysplastic forms present. Monocytes usually represent less than 10% of peripheral blood cells. The bone marrow is hypercellular, with an elevated myeloid-to-erythroid ratio, and marrow fibrosis may be prominent. In contrast to usual CML, basophilia is not prominent (usually <2% of peripheral blood white cells), the myeloid-to-erythroid ratio is usually less than 10 : 1, and no evidence of the Phila-delphia chromosome is noted by either routine karyotype or molecular studies. Although some abnormalities of gran-ulocyte nuclear lobation may be seen in CML, aCML usually has more typical dysplastic changes that may involve all cell lines (trilineage dysplasia). Granulocytes may show typical pseudo–Pelger-Huet changes and cytoplasmic hypogranu-larity. Dyserythropoiesis and megakaryocyte dysplasia are common and megakaryocytes may be reduced in number with associated thrombocytopenia. Percentages of bone marrow and peripheral blood blasts are less than 20%. aCML appears to be a more aggressive disease than usual CML, with progression occurring within 2 years.251,252 Patients may develop acute leukemia or may develop bone marrow failure secondary to marked fibrosis.

Cytogenetic and molecular genetic studies are essential in the diagnosis of aCML, to exclude t(9;22) or BCR/ABL1 of usual-type CML. No defining cytogenetic abnormality is known at this time for aCML, but del(20)(q11), trisomy 8 and 13, and i(17q) have been reported.247,252

Chronic Myelomonocytic Leukemia

CMML was originally defined as an MDS in the FAB classi-fication, but it has features similar to those of aCML and is

best classified as a mixed myeloproliferative syndrome/MDS. Patients often have both dysplastic changes and ele-vated white blood cell counts with splenomegaly. The disease has been divided into myelodysplastic and myelopro-liferative subtypes based on a white blood cell count of 13,000/µL or higher for the myeloproliferative and less than that number for the myelodysplastic types.249 Although the patients with the higher white blood cell counts have a higher incidence of splenomegaly, both types may demon-strate prominent dysplastic changes and the use of a white blood cell cutoff to separate the subtypes is somewhat arbi-trary.253 The diagnosis requires the presence of 1000/µL and >10% monocytes in the peripheral blood. The monocytes may be abnormal in appearance with bizarre nuclei and even cytoplasmic granules. Promonocytes, with more imma-ture nuclear chromatin, may be present in the blood, but monoblasts are usually not present or represent less than 2% of peripheral blood cells. The peripheral blood may demonstrate cytopenias and dysplastic changes more typical of the MDSs or dysplastic changes may be minimal. Although an elevated peripheral blood monocyte count is necessary for the diagnosis of CMML, such a diagnosis should not be made without examination of the bone marrow. Some cases of AML with monocytic blasts may show peripheral blood changes similar to those of CMML. The bone marrow of CMML is usually hypercellular and may demonstrate mono-cytic or granulocytic hyperplasia (Fig. 43-18). When granu-locytic hyperplasia is prominent, it may be difficult to distinguish the abnormal monocyte population from myelo-cytes. Erythroid precursors and megakaryocytes may have prominent dysplastic changes, but often these cell types appear normal. Ringed sideroblasts are present in increased numbers in some cases. Numbers of blast cells and pro-monocytes may be elevated, approaching 20%, and an elevation in blast cells is associated with a poorer prognosis.254,255

Ancillary studies are helpful in the differential diagnosis of CMML. Cytochemical testing for nonspecific esterase on the peripheral blood and bone marrow confirms the pres-ence of an increase in monocytes and can help differentiate abnormal monocytes of CMML from myelocytes in CML and aCML. Cytogenetic and molecular genetic studies, par-ticularly the absence of the Philadelphia chromosome and BCR/ABL1, are helpful in excluding CML. The most common cytogenetic abnormalities in CMML are trisomy 8, deletions of 7 or 7q, and abnormalities of 12p. Cases with the t(5;12)(q33;p13) involving the PDGFRBR and ETV6 genes,256 are usually associated with eosinophilia and are included in a separate category in the 2008 WHO classifica-tion.257,258 Mutations of RAS are detected in approximately one third of CMML cases.

The differential diagnosis between aCML and CMML may be difficult but is critical because of the worse progno-sis of patients with aCML when compared with CMML. CMML may be distinguished from aCML by peripheral blood features,249 but some overlap with aCML may occur (see Table 43-17). Monocytes are slightly elevated in aCML but do not usually exceed 10%, whereas the proportion of monocytes in CMML is greater than 10%. In addition, the degree of granulocyte dysplasia in CMML is not as pro-nounced as is usually seen in aCML. aCML demonstrates an increase in immature granulocytic cells, including blast cells, promyelocytes, and myelocytes, sometimes approach-

taBle 43-17Helpful Features in the Differential Diagnosis of Chronic Phase of Chronic Myelogenous Leukemia, Atypical Chronic Myeloid Leukemia, and Chronic Myelomonocytic Leukemia in Adults

CML aCML CMML

philadelphia chromosome/BCR/ABL1

+ − −

peripheral blood white blood cell count

+++ ++ +

peripheral blood basophils ≥2% <2% <2%peripheral blood monocytes <3% 3%-10% Usually >10%peripheral blood

promyelocytes, myelocytes, and metamyelocytes

>20% 10%-20% ≤10%

peripheral blood blasts ≤2% >2% <2%Granulocyte dysplasia −* ++ +Bone marrow erythroid

hyperplasia− − +

*Granulocyte dysplasia may be seen with accelerated phase of Cml.aCml, atypical chronic myeloid leukemia; Cml, chronic myelogenous leukemia;

Cmml, chronic myelomonocytic leukemia.

C


ing 20% in the peripheral blood; proportions of these cell types are almost always lower than 10% in the blood of patients with CMML.

Pediatric Myelodysplasia and Juvenile Myelomonocyte Leukemia

Children may develop any of the MDSs described for adults,259-262 although RARS is uncommon. These cases must be differentiated from known forms of AML with low blast counts, which include many of the AMLs with t(8;21) or inversion 16.263 These types of AML do not usually have associated dysplasia and should be considered acute leuke-mias and not myelodysplasias. Juvenile myelomonocytic leukemia and infantile monosomy 7 syndrome have also been described as pediatric MDSs, but they demonstrate features of both myelodysplasia and myeloproliferative neoplasms. Some investigators favor use of FAB criteria and terminology for these disorders,264 criteria that would place them under the diagnosis of CMML. Because these disorders appear to be clinically distinct from adult CMML, however, the WHO classification uses different terminology for these cases.

Juvenile myelomonocytic leukemia ( JMML), is rare, but it is the most common MDS/MPN of children.265 Children with JMML are more often boys (2:1 male-female ratio) younger than 14 years of age and develop the disease by the age 4 years in most cases. The children usually have eleva-tions of fetal hemoglobin and detectable I antigen. Skin lesions often precede the diagnosis and these children present with an elevated white blood cell count, of granu-locytes and monocytes, that may be identical to counts seen in CMML of adults. Thrombocytopenia is also often present and organomegaly is common. Dysplastic changes are often minimal and marrow hypercellularity occurs frequently. Overlap with adult aCML also occurs and the criteria for aCML and CMML are probably not appropriate in chil-dren.266 Proposed criteria for the diagnosis of JMML require clinical findings of hepatosplenomegaly, lymphadenopathy, pallor, fever, and rash.262 Proposed laboratory criteria are the absence of the Philadelphia chromosome, monocytosis greater than 1000/µL, and the presence of less than 20%

bone marrow blast cells. In contrast to adult CMML, JMML has an aggressive clinical course. Infantile monosomy 7 syn-drome267 is clinically similar to JMML and probably repre-sents a subgroup of JMML.268

Cytogenetic abnormalities other than monosomy 7, which may occur with any of the MDSs, are not specific for JMML. Similar to adult CMML, RAS mutations occur in approximately 20% of cases of JMML.269 An association also exists between JMML and neurofibromatosis type 1,261 and mutations of the NF1 gene may occur in up to 15% of patients without evidence of neurofibromatosis.269

Other Myelodysplastic/Myeloproliferative Neoplasms

Some cases demonstrate features of both myelodysplasia and myeloproliferative neoplasms and do not fit well into any of the previously mentioned categories. Many of these cases have typical features of myelodysplasia as well as an atypical finding more suggestive of a myeloproliferative neoplasms, such as marked marrow fibrosis and hypercellu-larity or organomegaly. Such cases may be termed MDS/MPN unclassifiable, with a comment describing the atypical findings. One such syndrome has features of RARS and thrombocythemia.247 These cases have no sex predilection or specific cytogenetic abnormality and must be differenti-ated from the 5q− syndrome myelodysplasias. A MDS/MPN neoplasm associated with isochromosome 17q has been described that occurs in adults with a male predominance and is associated with severe hyposegmentation of neutro-phil nuclei, monocytosis, and a high rate of transformation to AML.270 Many of these cases, however, meet criteria for CMML.

Plasma Cell Disorders

Multiple Myeloma

Multiple myeloma (plasma cell myeloma) is a clonal prolif-eration of plasma cells that usually express monoclonal immunoglobulin (Ig) G or A or monotypic κ or λ immu-noglobulin light chains without an associated heavy

Figure 43-18 n Chronic myelomonocytic leukemia. A, Numerous monocytes, including some with the appearance of myelocytes, are present in the bone marrow aspirate. B, The biopsy is hypercellular with abundant monocytes present.

A B

C

BoNe maRRow n 1561

chain.271,272 The disease usually affects older adults; reports in children or young adults are extremely uncommon.273 The disease occurs in men slightly more commonly than in women and in blacks more frequently than in whites. Multiple myeloma has different diagnostic clinical crite-ria.42,274-277 Most rely on the detection of a serum monoclo-nal paraprotein of at least 3.5 g/dL of IgG, 2 g/dL IgA, or 1 g/24 hours of light chains in the urine, radiologic detec-tion of bone lesions, and the presence of an increase in bone marrow plasma cells. Patients frequently have anemia and may have hypercalcemia. Although the monoclonal protein results in an elevated total serum protein, normal immuno-globulin levels are low. The detection of more than 30% monotypic plasma cells in the bone marrow is usually suf-ficient for the diagnosis, but the presence of 10% to 30% bone marrow plasma cells is often enough for a clinical diagnosis if other parameters, such as anemia, renal insuf-ficiency, or hypercalcemia, are present.

Patients with multiple myeloma characteristically have normocytic anemia. Thrombocytopenia and leukopenia are usually not present. Rouleaux formation of the red blood cells is common and is often associated with a blue back-ground on the smears. Both these findings are related to the elevated serum monoclonal protein. Circulating plasma cells are seen in some patients but are usually only a small number of white blood cells. The bone marrow is either normocellular or hypercellular (Fig. 43-19).278 Plasma cells counts are traditionally performed on aspirate material or imprints, but excellent results may also be obtained using CD138 staining of bone marrow biopsy material. The plasma cells vary in morphologic appearance and range from small, well-differentiated cells that are similar to normal plasma cells to poorly differentiated or plasmablas-tic forms with large central nucleoli or immature nuclear chromatin that may cause confusion with lymphoma cells or myeloblasts. Binucleated and trinucleated plasma cells are commonly identified in multiple myeloma. Cytoplasmic inclusions, including vacuoles, granules, and crystals, may occur in the plasma cells. Various morphologic grades of neoplastic plasma cells are described279,280 that center on the number of plasmablasts present. Plasmablasts are defined as having fine reticular nuclear chromatin with little or no chromatin clumping.281 The nucleus is enlarged and may

have a large nucleolus with a high nuclear-to-cytoplasmic ratio and minimal or no cytoplasmic hof. Some investiga-tors use a cutoff of less than 10% plasmablasts for well-dif-ferentiated disease and 50% or more plasmablasts as poorly differentiated disease.280 The presence of only 2% or more plasmablasts, however, correlates with a poor progno-sis.281,282 Other bone marrow elements are usually normal in appearance. Osteosclerosis is rarely seen in multiple myeloma283 and some cases are associated with the POEMS syndrome of polyneuropathy, organomegaly, endocrinop-athy, monoclonal gammopathy, and skin lesions.284

Multiple myeloma may be further subdivided into indo-lent and smoldering types.277 Indolent multiple myeloma is defined by the presence of more than 30% plasma cells with no clinical symptoms or associated disease features, often lower paraprotein levels (<7 g/dL IgG or <5 g/dL IgA), and three or fewer lytic bone lesions. Smoldering multiple myeloma has similar features, with between 10% and 30% plasma cells. Therefore, the plasma cell count is an essential element of the bone marrow report. The bone marrow cel-lularity and plasma cell count should be given for all speci-mens, and when possible a direct comparison between specimens and the previous marrow is recommended to properly evaluate response to therapy. Solitary plasmacy-toma differs from multiple myeloma by being a solitary soft tissue or bone lesion with no systemic symptoms of multi-ple myeloma and less than 10% plasma cell in the bone marrow. The term plasma cell leukemia is used when 20% or more of peripheral blood white cells are plasma cells, or an absolute plasma cell count of more than 2 × 109/L. This usually occurs with aggressive disease and is associated with short survival. These patients usually have anemia, thrombocytopenia, lymphadenopathy, and organomegaly. However, the detection of lower numbers of monotypic peripheral blood plasma cells is common in multiple myeloma and levels as low as 4% are associated with short-ened survival.285

Protein electrophoresis studies are recommended in all patients with suspected multiple myeloma,286,287 and the results of these studies coupled with the finding of more than 30% bone marrow plasma cells is often sufficient for diagnosis. However, when the differential diagnosis includes reactive plasmacytosis, immunophenotyping studies may

Figure 43-19 n Sheets of plasma cells are present in the bone marrow aspirate (A) and the biopsy (B) in a case of multiple myeloma.

A B

C


be extremely helpful. The detection of a monoclonal plasma cell population by flow cytometry requires detection of cytoplasmic light chains, testing not routinely performed in all laboratories. These studies are often coupled with detec-tion of CD38, an activation marker present on most plasma cells as well as T cells and other cell types, or CD138 (anti–syndecan-1), an antigen more specific for plasma cells.285,288 Paraffin section immunohistochemistry289 or in situ hybridization for the detection of immunoglobulin light chains is also often helpful for the determination of clonality as well as the detection of residual clonal disease. Although plasma cells represent the end-stage maturation of B cells, they frequently do not immunoreact with CD20, and the use of CD20 antibodies to evaluate plasma cell populations is unreliable. Although CD79a detects a higher percentage of plasma cells than does CD20, many cases also test negative for this antigen. Other paraffin section markers that are useful in multiple myeloma include CD138, which marks neoplastic and non-neoplastic plasma cells, and CD56, which is usually not expressed by non-neoplastic plasma cells.290 DNA labeling indices or proliferative rates are also used in some centers to evaluate the aggressiveness of multiple myeloma cases.291

Immunoglobulin heavy-chain gene rearrangements are present in multiple myeloma but are often more difficult to detect by PCR analysis than in other lymphoid malignant diseases.291 Approximately 40% of patients with multiple myeloma have cytogenetic abnormalities.292,293 Various cytogenetic translocations may be detected in multiple myeloma and often involve the immunoglobulin heavy-chain region of chromosome 14.294-296 Up to 30% of multiple myeloma cases demonstrate t(11;14)(q13;q32), which involves the BCL-1/cyclin D1 gene on chromosome 11 that is also translocated in mantle cell lymphoma, although the breakpoint sites within the cyclin D1 gene differ in multiple myeloma and mantle cell lymphoma.297 The t(4;14)(p16;q32) is present in approximately 15% of myelomas and involves the MMSET (multiple myeloma SET domain) or the FGFR3 gene of chromosome 4.298 Partial or complete deletions of chromosome 13 and abnormalities of chromosome 1 are the other most common cytogenetic findings in multiple myeloma. The chromosome 13 and 4p16 abnormalities are associated with a worse prognosis.292,299

Monoclonal Gammopathy of Undetermined Significance

Monoclonal gammopathy of undetermined significance (MGUS) is a relatively indolent disease that occurs in the absence of evidence of multiple myeloma, other chronic lymphoproliferative disorders, or amyloidosis.300 As in mul-tiple myeloma, it is more common in elderly and male patients and is the most common cause of a monoclonal serum protein. Patients with MGUS have a serum parapro-tein level of less than 3 g/dL, less than 10% bone marrow plasma cells, and no have anemia, renal failure, hypercalce-mia, or other clinical parameters of multiple myeloma. These patients do not require treatment, but 20% to 25% of cases eventually progress to myeloma, Waldenström’s mac-roglobulinemia, amyloidosis, or another chronic lympho-proliferative disorder.300,301 The only pathologic finding is

an increase of plasma cells of less than 10% in the bone marrow, but a recognizable plasma cell increase may not be present. The plasma cells are usually normal in appearance, and polyclonal plasma cells are usually admixed with the monoclonal cells.302 This mixture of polyclonal and mono-clonal cells may make interpretation of immunohistochemi-cal studies difficult, and such studies are not usually indicated.

Waldenström’s Macroglobulinemia

Waldenström’s macroglobulinemia, or primary macroglobulin-emia, is a clinical syndrome of an IgM monoclonal gam-mopathy, usually of 3 g/dL or more, that is associated with splenomegaly or hepatomegaly in 40% of cases.303-305 These patients are usually elderly with a slight male predomi-nance. In contrast to multiple myeloma, Waldenström’s macroglobulinemia is extremely unusual in black male patients. Patients have an increased risk of hemorrhage as well as a high frequency of hyperviscosity syndrome and cryoglobulinemia. This syndrome is characteristically asso-ciated with bone marrow and organ involvement by malig-nant lymphoma with plasmacytoid features, particularly lymphoplasmacytic lymphoma (Fig. 43-20).306,307

Normocytic anemia is the most common peripheral blood finding in Waldenström’s macroglobulinemia, and rouleaux formation with the characteristic blue background associated with an increase in paraprotein is also common. Peripheral blood involvement by the associated lympho-proliferative disorder is frequently present and may have the appearance of an increase in small lymphocytes, similar to chronic lymphocytic leukemia (CLL), or an increase in lymphoplasmacytic cells. Virtually any type of malignant lymphoma may involve the bone marrow in Waldenström’s macroglobulinemia, although lymphoplasmacytic lym-phoma is the most common type. The pattern may be nodular, interstitial, diffuse, or a mixture of more than one pattern. The lymphocytes are generally small on smears; some show eccentric, basophilic cytoplasm, similar to the cytoplasm of plasma cells, while often retaining the nuclear features of mature lymphocytes (plasmacytoid lympho-cytes). Some cases, however, demonstrate a frank plasma

Figure 43-20 n The bone marrow aspirate smear contains an increase in lymphocytes as well as obvious plasma cells in a case of lymphoplasma-cytic lymphoma that manifested as Waldenström’s macroglobulinemia.C

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cell component. Intranuclear inclusions in some of the lym-phoid cells (Dutcher bodies) are usually present and mast cells are usually increased in number.

Because any type of malignant lymphoma may cause clinical symptoms of Waldenström’s macroglo bulinemia, the bone marrow in these cases should be diagnosed as involved by the specific type of lymphoma with a comment acknowledging that the findings are consistent with the clinical setting of Waldenström’s macroglobulinemia.

Paraffin section immunohistochemistry is often helpful in identifying a monotypic plasma cell population in Waldenström’s macroglobulinemia that allows for the exclu-sion of a reactive lymphoplasmacytic population in the bone marrow. The detection of IgM heavy chains is helpful in excluding multiple myeloma, which usually expresses only IgA, IgG, or light chain and not IgM. Flow cytometry immunophenotyping may also be useful in identifying a monoclonal B-cell population and in the subclassification of the precise lymphoproliferative disorders. The lympho-plasmacytic lymphoma that is most commonly associated with Waldenström’s macroglobulinemia is CD5−. Even if morphologic evidence of peripheral blood involvement is not apparent, flow cytometry studies may still detect a small monotypic B-cell population. Gene rearrangement studies may be helpful, because all the malignant lymphomas asso-ciated with Waldenström’s macroglobulinemia are B-cell malignant diseases. No specific recurring cytogenetic abnor-malities are recognized in lymphoplasmacytic lymphoma. Although the t(9;14)(p13;q32) involving the immunoglob-ulin heavy-chain gene of chromosome 14 and the PAX5 gene of chromosome 9 were originally reported to be common with this disease,308 subsequent studies failed to confirm that finding.309,310

Heavy-Chain Diseases

The heavy-chain diseases are rare monoclonal proliferations expressing IgA, IgM, or IgG without an associated light chain. α Heavy-chain disease primarily involves the gastro-intestinal tract in young people, with an extensive lympho-plasmacytic or plasmacytic infiltrate and rarely involves the bone marrow.311 µ Heavy-chain disease usually has features identical to those of CLL, although vacuolated plasma cells have been described as a characteristic finding in the bone marrow.312 γ Heavy-chain disease may be associated with any type of malignant lymphoma and often produces a syn-drome similar to Waldenström’s macroglobulinemia with associated bone marrow involvement.313

Amyloidosis

Amyloidosis represents a heterogeneous group of disorders that can be divided into those derived from light-chain (usually λ)–derived fibrils (AL) or those caused by deposi-tion of amyloid protein A (AA). β2-microglobulin–derived and transthyretin (formerly known as prealbumin)–derived amyloid may also occur.314-316 AL amyloidosis is often termed primary, whereas AA amyloidosis is generally secondary disease. Clinically, amyloidosis can be divided into systemic and nonsystemic types, with the systemic type further divided into those cases associated with the following: plasma cell dyscrasias; reactive amyloidosis related to infec-

tions, autoimmune disorders, and malignant diseases; and familial, dialysis–associated, and senile systemic amyloido-sis. All types show the characteristic deposition of homoge-neous, acellular eosinophilic material that demonstrates apple-green birefringence when polarized on Congo red stain.

Most cases of primary amyloidosis are considered plasma cell dyscrasias with associated monoclonal gammopathy without evidence of multiple myeloma or Waldenström’s macroglobulinemia. Patients frequently have nephrotic syn-drome, congestive heart failure, orthostatic hypotension, or peripheral neuropathy.317 The bone marrow biopsy appears to be more reliable than is the aspirate smear in identifying amyloid deposits,318 but amyloid may be identifiable only in the bone marrow in one third of patients with known systemic disease.319 Amyloid deposition in the bone marrow may be in the form of sheets of the material, or deposits may be evident only in the walls of thickened blood vessels. Plasma cells are often increased in the bone marrow but may appear polyclonal in up to one third of cases despite the presence of a monoclonal serum protein.319 Bone marrow testing is often performed specifically to confirm or exclude the presence of amyloid deposits, which can usually be resolved with the Congo red stain.

Lymphoid Proliferations

Lymphocytic Leukemias of B Lineagechronic lymphocytic leukemia

CLL is a monoclonal B-cell proliferation of predominantly small lymphocytes involving the peripheral blood that is morphologically and immunophenotypically identical to small lymphocytic lymphoma (Fig. 43-21).320-322 CLL occurs more commonly in older adults with a male predominance and patients usually have generalized lymphadenopathy. The white blood cell count is normal or elevated with an absolute lymphocytosis. The diagnostic criteria for CLL are variable but generally require at least 5 × 109/L or more lymphocytes with at least 30% bone marrow small lympho-

Figure 43-21 n Chronic lymphocytic leukemia in the bone marrow char-acterized by a proliferation of small to medium-sized lymphocytes with clumped nuclear chromatin and scant cytoplasm. C


cytes.323 Bone marrow examination, however, is not required for this diagnosis. Demonstration of the characteristic immunophenotype of CLL is critical, and immunopheno-typing studies should be performed at diagnosis on all cases to exclude peripheral blood involvement by other lymphomas.

The peripheral blood shows predominantly small lym-phocytes with scant, basophilic cytoplasm and mature, clumped nuclear chromatin, but scattered large lymphoid cells and prolymphocytes with nucleoli may be present. Anemia and thrombocytopenia are usually not present, but when present they are associated with shortened survival; these parameters are included in the Rai and Binet staging systems.323,324 The bone marrow is almost always involved in CLL, and although the absolute number of small lym-phocytes identified on an aspirate smear has significance, the bone marrow biopsy pattern of infiltration is also impor-tant (Fig. 43-22).325 This pattern of infiltration may be nodular, interstitial, diffuse, or mixed. The pattern of infil-tration has prognostic significance, with the nodular pattern associated with the best prognosis and the diffuse pattern associated with a worse prognosis.326,327 As in the blood, the bone marrow lymphocytes are predominantly small, but large cells are commonly present in small numbers. A het-erogeneous pattern of small lymphocytes with admixed larger cells is a characteristic feature of CLL on histologic sections. Aggregates of large cells or prolymphocytes with prominent nucleoli in the bone marrow may reflect trans-formation to a higher-grade lymphoproliferative disorder,

but transformation of CLL is usually determined by periph-eral blood changes.

Morphologic variants of CLL can occur and include cases with cleaved cells, plasmacytoid cells, large cells, and increased prolymphocytes. The presence of large cells or prolymphocytes of between 10% and 55% of peripheral blood at presentation has been termed mixed cell type of CLL, mixed CLL/prolymphocytic leukemia (CLL/PLL), or CLL with increased prolymphocytes.328 Although such designa-tions may identify patients with slightly more aggressive disease, they are not made by all hematopathologists and are not part of the National Cancer Institute–Sponsored Working Group Guidelines for CLL.323 Cases with cleaved small lymphocytes or plasmacytoid cells that have the char-acteristic immunophenotype of CLL should be classified as CLL, but one small study of these cases suggests a more aggressive clinical course compared with more typical CLL.329 CLL may undergo prolymphocytoid transformation or Richter’s transformation. Prolymphocytoid transformation occurs when a patient with preexisting CLL develops more than 55% peripheral blood prolymphocytoid cells, which have more abundant cytoplasm than usual CLL lympho-cytes and have prominent nucleoli (Fig. 43-23).323,328 A paraimmunoblastic variant has been described in which the prolymphocytoid cells have a single prominent nucleo-lus that gives a similar appearance to immunoblast nuclei on histologic sections.330 Richter’s transformation refers to the development of any higher-grade lymphoma in a patient with CLL, but it usually represents development of

A B

C

Figure 43-22 n The major patterns of bone marrow infiltration by chronic lymphocytic leukemia are diffuse (A), nodular (B) and inter-stitial (C).

C

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large B-cell lymphoma. Transformation of CLL is generally associated with aggressive clinical behavior and short survival.331-333

Autoimmune disorders frequently occur in patients with CLL. These include autoimmune hemolytic anemia, idio-pathic thrombocytopenic purpura, red cell aplasia, and neu-tropenia.334,335 A careful search for erythroid precursors should be made on all CLL bone marrow specimens because red cell aplasia may be easily overlooked.

The term chronic lymphocytic leukemia is now reserved for cases of B-lineage neoplasms that have a characteristic immunophenotype.336-338 CLL demonstrates monoclonal immunoglobulin light and heavy chains, but this expres-sion, as well as the expression of CD20, is characteristically weak. These cells also express B-lineage–associated markers CD19 and CD79a and express CD23 and the T-cell–associ-ated markers CD5 and CD43. These cells usually test nega-tive for FMC7. Weak light-chain and CD20 expression and aberrant expression of CD5 are critical in the diagnosis of CLL. CD5-negative CLL is rare and peripheral blood involvement by other lymphoma types, such as marginal zone and follicular lymphomas, should be excluded. CLL cases with atypical immunophenotypes behave differently from usual-type CLL, often more aggressively, and are more often associated with splenomegaly.339,340 CD38 expression is also associated with more aggressive disease341 and often correlates with a lack of immunoglobulin heavy-chain (IGH) gene mutations in CLL (see later). ZAP-70 expres-sion, detected by either flow cytometry or immunohisto-chemistry, has been shown to correlate with a lack of IGH mutation and a worse prognosis in CLL.342,343 Bright light-chain expression and loss of CD5 commonly occur with prolymphocytoid transformation of CLL.

Gene rearrangement studies enable one to identify an immunoglobulin heavy-chain rearrangement in virtually all cases of CLL. Lack of mutation of the variable region of IGH has been shown to be associated with more aggressive disease and atypical morphologic features.341,344 Thus, CLL can be divided into naïve (unmutated) and mutated types by these molecular studies or by evaluation of ZAP-70

expression. Cytogenetic abnormalities, other than IGH rearrangements and mutations, occur in approximately 80% of cases of CLL but can usually only be detected by FISH. In order of frequency, deletions of chromosome band 13q14, 11q23 deletions, trisomy 12, 6q21 deletions, and deletions or mutations of the p53 gene on 17p are the most common cytogenetic abnormalities in CLL.345-349 Deletions of 13q confer the best prognosis, 17p and 11q deletions have the worst prognosis, and the remaining are intermediate in prognosis. Trisomy 12 is usually restricted to cases with atypical features, such as mixed CLL/PLL morphology, bright surface immunoglobulin expression, or transforma-tion to a higher grade process. The combined use of IGH mutation results and detection of karyotype abnormalities using FISH analysis seems to be the best means of predict-ing outcome in CLL patients.350,351

b-cell prolymphocytic leukemia

B-cell prolymphocytic leukemia (B-PLL) occurs in older patients with no history of CLL and has a male predomi-nance. Patients usually have splenomegaly without periph-eral lymphadenopathy and a markedly elevated white blood cell count.352 The peripheral blood contains at least 55% prolymphocytes, but this disease differs from prolympho-cytoid transformation of CLL by the lack of preexisting CLL.328 Anemia and thrombocytopenia are commonly present. The bone marrow is usually extensively involved in an interstitial, diffuse, or mixed pattern by small and medium-sized lymphocytes with prominent nucleoli (Fig. 43-24).353 The nodular bone marrow pattern that is often seen in CLL is uncommon in B-PLL. The medium-sized cells tend to have clearing of nuclear chromatin and mitotic figures are frequent. Admixed larger cells are also common. The cells are monotypic B cells with bright immunoglobulin light-chain expression and variable CD5 expression. They usually test positive for FMC7. Immunoglobulin heavy-chain gene rearrangements are usually detectable and cyto-genetic abnormalities similar to CLL may be identified.354 Mutations of p53 are reported to be much more common in B-PLL than in other B-cell malignant diseases.355

The differential diagnosis of B-PLL includes T-cell pro-lymphocytic leukemia (T-PLL), mixed CLL/PLL, prolym-

Figure 43-23 n Chronic lymphocytic leukemia with increased prolym-phocytes. Note the increased numbers of bone marrow cells with moder-ately abundant cytoplasm and nucleoli. The presence of more than 55% of such cells in the peripheral blood is characteristic of prolymphocytoid transformation.

Figure 43-24 n B-cell prolymphocytic leukemia is characterized by cells with abundant cytoplasm and a prominent central nucleolus. C


phocytoid transformation of CLL, blood and bone marrow involvement by large cell lymphoma, marginal zone lym-phoma and mantle cell lymphoma, and even acute leuke-mia. The clinical history helps to exclude transformation of CLL or large cell lymphoma in most cases and the prolym-phocyte count distinguishes mixed CLL/PLL (≤55% periph-eral blood prolymphocytes) from B-PLL (>55% peripheral blood prolymphocytes). Splenic marginal zone lymphoma may have a similar clinical presentation but does not usually have a high percentage of cells with a prominent central nucleolus. Mantle cell lymphoma involving the peripheral blood and bone marrow may be difficult to distinguish from PLL, and some cases may demonstrate the characteristic single, central nucleolus of PLL.356 Cytogenetic or molecu-lar genetic studies for t(11;14) of mantle cell lymphoma or immunophenotypic studies for cyclin D1/Bcl-1 are helpful in identifying cases of mantle cell lymphoma (see later). Immunophenotyping studies easily distinguish B-PLL from a T-cell malignant disease or acute leukemia.

hairy cell leukemia

HCL is a rare monoclonal B-cell disorder that usually affects elderly men in the sixth decade of life.357,358 Pancytopenia and splenomegaly without associated lymphadenopathy are the common clinical presentations of these patients. The anemia is usually normocytic and normochromic. Monocy-topenia is almost always present. Abnormal lymphocytes

are usually scant in the peripheral blood but have cytoplas-mic projections or “hairs” (Fig. 43-25). This feature is non-specific and villous cytoplasmic projections of lymphocytes may be seen with other lymphoproliferative disorders. The abnormal cell population has slightly basophilic cytoplasm and the cell nucleus is round, slightly indented, or reniform. The nuclear chromatin is smooth without the characteristic chromatin clumping seen in CLL or normal small lympho-cytes. Nucleoli are indistinct or absent in the hairy cells. Platelets are usually decreased in number. The bone marrow is almost always involved in HCL, but the pattern of involve-ment is interstitial and may be subtle on H&E-stained sec-tions. The bone marrow also demonstrates fine reticulin fibrosis, often making aspiration difficult. Because of the limited material available by aspiration in many cases, bone marrow biopsy is often necessary for the diagnosis.359 The bone marrow may be normocellular or hypercellular, but normal hematopoietic elements are commonly reduced in number. Small lymphocytes, similar to those seen in the blood, are identified on aspirate smears or touch imprints. The bone marrow biopsy shows an interstitial infiltration by small cells with moderately abundant clear to slightly eosinophilic cytoplasm without nucleoli. The cells with oval or reniform nuclei and abundant cytoplasm often have a “fried-egg” appearance. When the cells are admixed with other bone marrow cells they may be mistaken for erythroid precursors.

Figure 43-25 n Hairy cell leukemia. A, The bone marrow aspirate is often paucicellular but may contain cells with reniform nuclei and moderately abun-dant cytoplasm that is often friable and gives a hairy appearance. B, The bone marrow biopsy shows an interstitial infiltrate of small cells with clear cytoplasm. The cells stain for CD20 (C) and tartrate-resistant acid phosphatase (TRAP) (D) by immunohistochemistry.

A B

C D

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HCL variant is a chronic lymphoproliferative disorder that has features of HCL and B-PLL.360 The white blood cell count is usually elevated, as in B-PLL. The lymphoid cells have cytoplasmic projections, similar to HCL, but also dem-onstrate a prominent central nucleolus, similar to B-PLL (Fig. 43-26).

Immunophenotyping studies performed on peripheral blood or bone marrow in HCL demonstrate a CD5-negative monoclonal B-cell population. The cells usually test strongly positive for CD11c, CD25, and CD103 and demonstrate bright surface immunoglobulin heavy and light chains and bright CD20.361-363 A subgroup of cases expresses CD10.364 By cytochemical methods, the cells test positive for TRAP.19 Because of the high frequency of dry taps in patients with HCL, immunohistochemical studies on bone marrow biopsy material may be necessary.365 The interstitial small lympho-cyte infiltrate immunoreacts for CD20 and confirms the B lineage of the cells. Results of testing for DBA.44 and annexin are also positive on the hairy cells but negative on the cells of CLL.366 Hairy cells do not aberrantly coexpress CD43 but may test weakly positive for cyclin D1.367 A paraf-fin section antibody directed against TRAP is also available (see Fig. 43-25D),368 and the combined expression of TRAP and DBA.44 is reported to be fairly specific for HCL.369 Cytogenetic studies are usually not helpful because of limited growth of the specimens. Although no specific abnormality has been described for HCL, clonal abnormali-ties involving chromosome 5, including trisomy 5, are reported in 40% of cases.370 Immunoglobulin heavy-chain gene rearrangements are consistently detected in HCL.

The differential diagnosis of HCL in the blood includes splenic marginal zone cell lymphoma/splenic lymphoma with villous lymphocytes (SLVL), HCL variant, and other chronic lymphoproliferative disorders, including CLL. Immunophenotyping easily excludes CLL and mantle cell lymphoma by the lack of CD5 expression in HCL. Overlap exists between the immunophenotype of SLVL and HCL, although SLVL is more frequently CD103 negative and is usually TRAP negative. The pattern of bone marrow infiltra-tion differs between HCL and most lymphomas. In general, lymphomas form sinusoidal aggregates, nodules, or sheets in the bone marrow, in contrast to the interstitial pattern of

bone marrow involvement by HCL. CD25 expression is reportedly less common in HCL variant than in usual-type HCL,371 but the morphologic feature of a prominent nucleo-lus in HCL variant is the primary means of differentiating these two diseases.

T-Cell Leukemiast-cell prolymphocytic leukemia

T-PLL is a clonal T-cell proliferation that occurs most com-monly in elderly patients and has a slight male predomi-nance.328,372,373 The disease also occurs frequently in younger patients with ataxia telangiectasia.374 Patients have a mark-edly elevated white blood cell count as well as organomeg-aly and lymphadenopathy. Nodular or maculopapular skin lesions are also common. The peripheral blood white blood cell count is usually greater than 100 × 109/L with a pre-dominance of medium-sized cells with abundant basophilic cytoplasm and a single prominent nucleolus (Fig. 43-27). These cells are similar to B-cell prolymphocytes but may have a more convoluted nucleus than in B-PLL. Normocytic anemia and thrombocytopenia are common. The bone marrow may not be involved to the degree that would be expected by the marked elevation in peripheral blood pro-lymphocytes. The pattern of involvement may be intersti-tial, diffuse, or mixed and reticulin fibrosis is frequently present (Fig. 43-28).353 In general, T-PLL is an aggressive disease with short survival. However, a subpopulation of patients with T-PLL, including many with ataxia telangiec-tasia, have an initial, indolent disease course that eventually transforms to the more typical aggressive disease.375

Immunophenotyping is necessary to distinguish T-PLL from B-PLL and is often helpful in excluding acute leuke-mia. T cell-associated antigens CD2, CD3, CD5, and CD7 are expressed by T-PLL and surface CD3 is present. Most cases are CD4+, but a subset of cases expresses CD8 or both CD4 and CD8. The absence of both CD20 and immuno-globulin light-chain expression excludes B-PLL. The lack of TdT and CD1a expression and the presence of surface CD3 exclude most cases of T-cell ALL. T-cell receptor gene rear-rangements are uniformly detectable in T-PLL. Cytogenetic

Figure 43-26 n The peripheral blood cells of hairy cell leukemia variant have the friable cytoplasm typical of hairy cell leukemia and the prominent nucleolus of prolymphocytic leukemia.

Figure 43-27 n T-cell prolymphocytic leukemia in the peripheral blood is indistinguishable from B-cell prolymphocytic leukemia with abundant cytoplasm and a prominent central nucleolus. C


abnormalities in T-PLL include inv(14)(q11q32) and t(14;14)(q11;q32), involving the TCL1 gene in the region of the T-cell receptor α/β locus, iso(8q), trisomy 8, 12p13 deletions, and t(X;14)(q28;q11).375,376 Abnormalities of chromosome region 11q22-23, involving the ATM tumor suppressor gene that is consistently mutated in ataxia tel-angiectasia are present in some patients with T-PLL even in the absence of ataxia telangiectasia.377

Some T-cell chronic lymphoproliferative disorders have cells with morphologic features similar to those of B-CLL without the prominent nucleolus typical of usual-type PLL.378,379 Cases of this type are considered small cell vari-ants of T-PLL, and the term T-cell CLL should no longer be used. Although the median age and white blood cell count are lower in these patients than in usual-type T-PLL, these cases have immunophenotypic and cytogenetic features similar to those of T-PLL and a similarly aggressive clinical course.

large granular lymphocytosis

Elevations of the number of peripheral blood large granular lymphocytes (LGLs) may be reactive, such as seen follow-ing viral infections, or may be clonal neoplasms of T cells or NK cells. Patients with prolonged elevations of LGLs with associated neutropenia frequently have the clonal disease of large granular lymphocytic leukemia.380-384 These patients are usually elderly, may have associated infections or a previously diagnosed autoimmune disorder, and may have mild to moderate splenomegaly, but they rarely have lymphadenopathy.

The peripheral blood white blood cell count is normal to slightly elevated and neutrophils are usually decreased in number. LGLs predominate, with counts usually higher than 2 × 109/L for 6 months or more. The cells have clear to pale-staining cytoplasm with multiple azurophilic cyto-plasmic granules (Fig. 43-29) and are medium sized to large with mature round to indented nuclei without prominent nucleoli. Anemia and thrombocytopenia are present in some patients. Rouleaux formation may be present, related to a polyclonal hypergammaglobulinemia that occurs in some of these patients. The bone marrow is usually slightly hypercellular with a mild interstitial infiltration of LGLs or small non-paratrabecular aggregates that may be difficult to

identify on H&E-stained sections.385 Other hematopoietic elements are normal or may be slightly decreased in number.

LGL leukemia is further subdivided into T-cell and NK cell types. The T-cell proliferations express CD2, surface and cytoplasmic CD3, and CD8, but they may also express NK-associated markers such as CD16, CD56, and CD57. The T-cell type of LGL is the most common, accounting for approximately 85% of cases. These cases have T-cell receptor gene rearrangements, a finding that is helpful in excluding a reactive T-cell LGL proliferation.386 Immuno-histochemical studies of bone marrow biopsy material may also be useful in the differential diagnosis of reactive versus neoplastic T LGLs. The detection of clusters of CD8+ T cells correlates more with T-cell receptor gene rearrangements, whereas a lack of such cells in clusters is more common in reactive proliferations (Fig. 43-30).387 The NK cell type of LGL leukemia tests negative for surface CD3 but may express CD8 and usually tests positive for CD16 and CD56. These cases do not demonstrate T-cell receptor gene rear-rangements and require prolonged evidence of the prolifera-tion for a diagnosis of leukemia. NK proliferations that are associated with leukocytosis greater than 11 × 109/L are more likely to be associated with persistent disease.388 T-

Figure 43-28 n The bone marrow biopsy of T-cell prolymphocytic leukemia shows a diffuse infiltration of lymphoid cells (A) and an increase in reticulin fibrosis (B).

A B

Figure 43-29 n The lymphocytes of large granular lymphocytosis have abundant cytoplasm with a variable number of cytoplasmic granules.C

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LGL leukemia is usually an indolent disease with an increase in infections secondary to the associated neutropenia, but T-cell LGL with CD56 expression may behave more aggres-sively.389 NK LGL leukemia is less common than is the T-cell type, but some reports indicate a more aggressive clinical course. Because reactive LGL proliferations are indistin-guishable from LGL leukemia, gene rearrangement studies are essential in the T-cell proliferations and evidence of prolonged elevations in NK cell proliferations are necessary before a definite diagnosis can be made. In rare aggressive NK cell leukemias, cells may resemble myeloblasts and the disease has an aggressive clinical course.390 These cases are uncommon but should be distinguished from both mature LGL leukemias and AMLs with CD56 expression.

In contrast to some of the other T-cell and NK/T-cell lymphoproliferative disorders, LGL leukemias test negative for Epstein-Barr virus (EBV) and for human T-cell lym-phoma virus (HTLV)-1. Cytogenetic abnormalities are not usually detected in LGL leukemia.

adult t-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a clonal T-cell proliferation associated with HTLV-1 infection.328,391 This infection is more common in southwest Japan, West Africa, the Caribbean, and the southeast United States. It is rare in other locations. Most patients are adults with lymphade-nopathy, with or without organomegaly. Skin lesions and hypercalcemia are also common. Approximately one third of patients will present without lymphadenopathy but will have 10% or more abnormal cells in the blood and are con-sidered leukemic. The remaining patients present with lym-phoma or a mixture of leukemia and lymphoma.

The peripheral blood shows a marked elevation in the white blood cell count resulting from leukemic cells. The abnormal lymphocytes are medium sized and have a char-acteristic hyperlobated, flower-like nucleus or a multilo-bated knobby nucleus with moderate to scant, slightly basophilic cytoplasm. Hyperlobated nuclei may be rare or absent in some cases, with small, uniform, CLL-like cells seen more commonly in the chronic or smoldering form of the disease.392 The bone marrow is frequently normocellular with minimal and focal interstitial or diffuse involvement

seen in approximately half of cases.391,393 This bone marrow involvement is usually non-paratrabecular and may be subtle even with extensive peripheral blood involvement. Patients with hypercalcemia commonly show prominent osteoclastic activity and bone resorption. Acute, chronic, lymphomatous, and smoldering types of ATLL are described.328,394 In the acute form, many atypical lympho-cytes with abnormal nuclear lobations are present in the blood. In the chronic form, the lymphoid population is more uniform with only rare nuclear folds or bilobated forms present. The lymphomatous form does not involve the blood and manifests with adenopathy. Smoldering ATLL is characterized by up to 3% abnormal peripheral blood cells with changes similar to those seen in the chronic form of the disease. The acute and lymphomatous forms are aggressive diseases, whereas the chronic and smoldering forms of ATLL are more indolent diseases that may progress to an acute disease.

The leukemic cells of ATLL express CD2, surface CD3, and CD5, are usually CD4+ and CD25+, frequently do not express CD7 or CD8, and lack TdT. The CD25 expression differs from many of the other chronic T-cell proliferations. The lack of CD7 in ATLL is considered useful in the differ-ential diagnosis of T-PLL, which is usually CD7+.395 The detection of HTLV-1 and T-cell receptor gene rearrange-ments is helpful in establishing the diagnosis of ATLL. Cytogenetic abnormalities, including multiple trisomies, loss of sex chromosomes, translocations involving 14q, and deletions of 6q, have all been reported in ATLL.396

sézary’s syndrome and other t-cell proliferations

Other T-cell lymphomas may involve the blood and bone marrow and may produce a leukemic peripheral blood picture. These lymphomas are usually easily recognized and diagnosed if the appropriate clinical information of previ-ous lymphoma or clinical evidence of mycosis fungoides is relayed to the pathologist. Sézary’s syndrome is a leukemic manifestation of mycosis fungoides that occurs most com-monly in middle-aged men.397 Patients have the character-istic generalized erythroderma of mycosis fungoides and may have lymphadenopathy, organomegaly, and other der-matologic problems. The peripheral blood shows an ele-

Figure 43-30 n Although the lymphoid infiltrate of large granular lymphocytic leukemia may be subtle, results of immunohistochemical studies often show clusters of CD3+ (A) and CD8+ (B) lymphocytes. The presence of such clusters tends to correlate with the presence of a T-cell receptor gene rear-rangement.

A B

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vated white blood cell count resulting from circulating Sézary cells. These cells may be subdivided into large- and small-cell types.398,399 The large-cell type should be present for diagnosis because small Sézary cells may be identified in patients with non-neoplastic dermatosis. The Sézary cell has scant to moderately abundant pale or slightly basophilic cytoplasm that may contain vacuoles. The nucleus is enlarged with an irregular, cerebriform appearance with multiple nuclear indentations. The nuclear chromatin may be focally dense, or hyperchromatic, and nucleoli are not prominent (Fig. 43-31). The small-cell variant has dense chromatin and less conspicuous nuclear indentations. The bone marrow is usually normocellular with subtle focal nodular or interstitial involvement by small irregular lym-phocytes or may be uninvolved.400,401 Large transformed cells may be admixed with the small irregular cells. Aggre-gates of cytologically normal lymphocytes should not be overinterpreted as definite evidence of bone marrow involve-ment. Bone marrow eosinophilia may be present even in the absence of bone marrow involvement by lymphoma.

Immunophenotyping demonstrates a mature T-cell immunophenotype with surface CD3 expression and the Sézary cells are usually CD4+ and CD8−. Although both Sézary cells and the cells of ATLL are CD4+ T cells with irregular nuclear contours, they differ by the lack of flower-like nuclei, hypercalcemia, evidence of HTLV-1, and CD25 expression in most cases of Sézary’s syndrome. T-cell recep-tor gene rearrangements are present in Sézary’s syndrome and may be useful in excluding reactive T-cell prolifera-tions. Gene rearrangement studies, however, must be cor-related with other pathologic and clinical findings because T-cell receptor gene rearrangements have been reported in the peripheral blood of some patients with benign inflam-matory skin lesion.402 Clonal cytogenetic abnormalities are present in the peripheral blood of over half of patients with Sézary’s syndrome, but a wide variation exists in the specific types of abnormalities.403

The term Sézary cell leukemia has been used to describe clonal peripheral blood T-cell disorders with convoluted cell nuclei similar to those seen in Sézary’s syndrome.404,405 In contrast to Sézary’s syndrome, these patients do not have erythema or other evidence of skin involvement at presenta-tion. Immunophenotypic and cytogenetic analyses of these

cases suggest that they represent morphologic variants of T-PLL.

Lymphomas

Any type of B- or T-lineage malignant lymphoma may involve the peripheral blood and bone marrow.406-408 Details of the different lymphoma types are described in Chapter 41, and not all lymphoma types are covered in this chapter. A primary diagnosis of malignant lymphoma can be made on bone marrow material using criteria similar to those used on lymph nodes. Immunophenotyping studies are often useful in confirming the presence of lymphoma and in subclassifying the process.338,409 The evaluation of the bone marrow in patients with a known diagnosis of malig-nant lymphoma is critical in the staging of the disease. When the bone marrow is involved by lymphoma, specific information that should be mentioned in the report includes the type of lymphoma, pattern of involvement, and percent-age of involvement in terms of percentage of all nucleated marrow cells.410 Discordance between bone marrow and lymph node lymphoma type is common, with lower grade lymphoma often identified in the bone marrow of patients with higher grade lymphoma elsewhere.411,412 Because the detection of low-grade lymphoma in the bone marrow of a patient with a history of intermediate-grade or high-grade lymphoma confers a worse prognosis,413 a small lymphocyte proliferation in the bone marrow should not be automati-cally assumed to be evidence of lack of involvement in a patient with an intermediate-grade or high-grade lymphoma elsewhere and a designation of atypical lymphoid aggregate may be necessary in some cases (see later).

Immunohistochemical, flow cytometry immunopheno-typing, and gene rearrangement studies are all useful in the primary diagnosis of peripheral blood and bone marrow involvement by lymphoma.414 Table 43-18 summarizes some of the most useful features of various lymphomas and other B-cell lymphoproliferative disorders in the blood and marrow. Flow cytometry is of less value in the evaluation of residual disease in the bone marrow and results are often negative when the morphologic or immunohistochemical evaluation identifies evidence of residual disease.415 Newer flow cytometry strategies, including CD19 gating, will cer-tainly improve this method for detecting residual disease. Gene rearrangement studies are helpful in the evaluation of residual disease, but detection of very low-level disease requires tumor-specific primers made from the patient’s original lymphoma specimen, methods not currently offered in most institutions.416 PCR testing for specific transloca-tions, such as t(14;18) of follicular lymphoma or t(11;14) of mantle cell lymphoma, detects much lower amounts of residual disease without the expense of tumor-specific primers.

follicular lymphoma

Follicular lymphoma in the peripheral blood may mimic other chronic lymphoproliferative disorders. Cells with cleaved nuclei are usually identifiable, but subclassification of lymphoproliferative disorders by peripheral blood mor-phologic features is often difficult. Follicular lymphomas usually involve the bone marrow in a paratrabecular pattern (Fig. 43-32), although a diffuse pattern of involvement that

Figure 43-31 n In Sézary’s syndrome, the peripheral blood cells have dense nuclear chromatin and many have irregular, cerebriform nuclei.

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includes paratrabecular disease may be present with exten-sive bone marrow disease.406-408 An increase in reticulin fibrosis in association with the lymphoid aggregates is common and often limits the ability to aspirate lymphoma cells for smears or immunophenotyping studies. Small col-lections of lymphoid cells immediately adjacent to bone trabeculae without intervening marrow or adipose tissue should be considered as evidence of bone marrow involve-ment in patients with known follicular lymphoma and should be considered highly suggestive of lymphoma in patients without known lymphoma. These aggregates contain a mixture of small cells with irregular nuclei and a variable number of large lymphoid cells. Predominantly small cleaved cell lymphoma (grade 1 of the WHO classifi-cation) is the most common type of follicular lymphoma seen in the bone marrow.

Immunophenotyping of peripheral blood or bone marrow suspected of being involved by follicular lymphoma can be extremely helpful.338 The lymphoid cells are monoclonal B

cells that often express CD10 but do not express CD5. Absence of CD10 expression, however, does not exclude the diagnosis of follicular lymphoma. Immunophenotyping of bone marrow specimens looking for focal or residual involvement by known follicular lymphoma is less helpful. Results of the bone marrow biopsy are frequently positive even when flow cytometry immunophenotyping does not detect a clonal B-cell population. In patients with known follicular lymphoma and characteristic paratrabecular lym-phoid aggregates in the bone marrow, paraffin section immunophenotyping is not necessary for a diagnosis of bone marrow involvement and may actually cause unneces-sary confusion when only small aggregates are present. Fol-licular lymphomas characteristically have large numbers of associated, non-neoplastic T cells, and a small paratrabecu-lar lymphoid aggregate may show a mixture of T and B cells by immunohistochemistry that could incorrectly suggest a reactive aggregate. In patients without a history of lymphoma and large aggregates in the bone marrow, paraffin section immunohistochemistry may be helpful. The B cells frequently test positive for CD10 and bcl-2 protein. Molecular genetic or cytogenetic studies may be helpful in confirming a diagnosis of follicular lymphoma involving the blood or bone marrow. The immunoglobulin heavy-chain gene rearrangement of a significant number of follicular lymphomas cannot be detected using the PCR method. Most follicular lymphomas demonstrate t(14;18)(q32;q21) involving the immunoglobulin heavy-chain gene on chromosome 14 and the BCL2 gene on chro-mosome 18,417 which can be detected by PCR analysis in most cases.418,419

mantle cell lymphoma

Mantle cell lymphoma also frequently involves the periph-eral blood and bone marrow.407,420-422 In the blood and bone marrow, an increase in small to medium-sized lymphocytes is often present. These lymphocytes vary from cells with round nuclei with small nuclear chromocenters to cells with irregular and cleaved nuclei. A separate population of prolymphocytes is not seen, but the leukemic cell popula-tion may mimic de novo PLL. The biopsy may be normocel-

taBle 43-18Immunophenotypic Findings of Peripheral Blood and Bone Marrow B-Cell Lymphoid Proliferations

CLL PLL MCL FCCL HCL MZL/SLVL

Cd19 + + + + + +Cd20 weak +/− + + + + +light chains weak +/− + + + + +Cd5 + −/+ + − − −Cd10 − − −/+ +/− −/+ −Cd23 + −/+ − +/− − −Cd43 + −/+ + − − −*Cd103 − − − − + −/+Cyclin d1 − − + − weak +/− −dBa.44/tRap − − − − + −FmC7 − + + −/+ + +

*approximately 40% of nonsplenic marginal zone cell lymphomas are Cd43+.

Figure 43-32 n Follicular lymphoma preferentially involves the bone marrow in a paratrabecular pattern. Any lymphoid aggregate of the bone marrow in this location should be considered highly suspicious for lym-phoma, and this pattern in a patient with a history of follicular lymphoma is diagnostic of bone marrow involvement by the disease.

+, positive; +/−, usually positive; −/+, usually negative; −, negative; Cll, chronic lymphocytic leukemia; FCCl, follicular center cell lymphoma; HCl, hairy cell leukemia; mCl, mantle cell lymphoma; mZl/SlVl, marginal zone lymphoma/splenic lymphoma with circulating villous lymphocytes; pll, B-cell prolymphocytic leukemia.

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lular or hypercellular with nodular, interstitial, paratrabecular, diffuse, and mixed patterns of involvement. A mixed nodular and paratrabecular pattern is common in this disease. The lymphoid cells are homogeneous in size with generally scant cytoplasm and small nuclear chromocenters. Epithe-lioid histiocytes are often admixed with the lymphoma cells, but admixed large lymphoid cells are not a feature of mantle cell lymphoma. The blastic type of mantle cell lym-phoma has cells with fine nuclear chromatin that may be mistaken for lymphoblastic leukemia/lymphoma in the presence of a diffuse pattern of marrow involvement. The immunophenotypic findings are useful in excluding a lym-phoblastic process.

Immunophenotyping of the blood and bone marrow show a monoclonal B-cell population with bright light-chain and CD20 expression, in contrast to CLL, and the cells characteristically express CD5, CD43, and FMC7 without expression of CD23. In contrast to lymphoblastic malignancies, blastic mantle cell lymphoma tests negative for TdT. The cells show nuclear expression of cyclin D1/ bcl-1.423,424 The detection of CD5, FMC7, and cyclin D1 on a monoclonal B-cell population is specific for this disease. If the diagnosis of mantle cell lymphoma is already estab-lished, all these studies may not be necessary. Demonstra-tion of a predominance of B cells with aberrant coexpression of CD5 or CD43 in a questionable lymphoid infiltrate is usually sufficient for the confirmation of bone marrow involvement by previously diagnosed disease. Immuno-globulin heavy-chain gene rearrangements are usually easily detectable in mantle cell lymphoma by PCR analysis. The t(11;14)(q13;q32), involving the immunoglobulin heavy-chain gene on chromosome 14 and the BCL1 gene on chromosome 11, is present in most cases of mantle cell lymphoma.425 The PCR test most commonly used for this translocation, however, only detects the major translocation cluster of t(11;14) and results are positive in only 40% to 50% of cases.426 FISH methods detect a much higher per-centage of cases, and this method is preferred over PCR for a primary diagnosis.

other non-hodgkin’s lymphomas

Bone marrow involvement by small lymphocytic lymphoma and lymphoblastic lymphoma is similar to that already

described for CLL and ALL. Morphologic and immunophe-notypic features similar to those used in lymph nodes and other sites may be used in the diagnosis of bone marrow involvement by other lymphoma types, including large cell lymphoma (see Chapter 41). Some patients without a history of lymphoma show bone marrow involvement by small lymphoid cells that are predominantly of B lineage but do not have diagnostic immunophenotypic features of CLL/small lymphocytic lymphoma, mantle cell lymphoma, or follicular lymphoma. Such cases are most often marginal zone lymphomas, but they may also represent lymphoplas-macytic lymphomas or CD10-negative follicular lympho-mas. Further workup of these patients often reveals evidence of lymphoma at other sites that is more suitable for classification.

hodgkin’s disease

Although eosinophilia is common in Hodgkin’s disease, peripheral blood involvement by neoplastic cells is not. The bone marrow is most frequently involved in patients with a known diagnosis of Hodgkin’s disease, but a primary diag-nosis of the disease can be made on a bone marrow speci-men. Any type of Hodgkin’s disease may involve the marrow, although involvement by the lymphocyte predominance type is uncommon.427 Bone marrow eosinophilia, lympho-histiocytic aggregates, and granulomas all may occur in patients with Hodgkin’s disease, and their presence does not definitely indicate the presence of bone marrow involve-ment by the disease.428 Identification of Reed-Sternberg cells or their mononuclear variants, usually in association with marrow fibrosis, is required for the diagnosis (Fig. 43-33). These cells are large, may be multinucleated, and have large eosinophilic nucleoli. Diagnostic cells are usually not easily identified on bone marrow aspirate smears. Speci-mens showing large, atypical lymphohistiocytic aggregates without obvious Hodgkin cells should have multiple H&E-stained levels cut through the tissue to exclude the presence of disease. Immunohistochemical studies on the biopsy material are helpful in confirming the presence of mono-nuclear Reed-Sternberg cell variants. These cells should be CD45−, CD15+, and CD30+.336 This immunophenotype is typical of classic Hodgkin’s disease, including nodular scle-rosis, mixed cellularity, and lymphocyte-depleted types.

Figure 43-33 n Hodgkin’s disease involves the bone marrow with patchy areas of associated fibrosis (A). The fibrous areas must contain classic Reed-Sternberg cells or their mononuclear variants (B).

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CD15 expression by normal hematopoietic cells must not be overinterpreted as a positive result, and expression in neoplastic cells of the marrow may be perinuclear and weak. The background small lymphocytes are CD3+ T cells. CD20 expression by large neoplastic cells may be seen in any type of Hodgkin’s disease, but strong, uniform staining for CD20 with CD45 expression and a lack of CD15 or CD30 expres-sion comprise the characteristic immunophenotype of nodular lymphocyte predominance (L and H) Hodgkin’s disease. In the bone marrow, however, it is usually not possible to distinguish nodular L and H Hodgkin’s disease from bone marrow involvement by T-cell–rich large B-cell lymphoma. Subclassification of the types of classic Hodgkin’s disease is also not possible on bone marrow material alone.

Other than immunohistochemistry, ancillary studies are of limited value in Hodgkin’s disease. This disorder is usually not detectable by flow cytometry studies. In situ hybridization or immunohistochemical testing for EBV is positive in Hodgkin cells in approximately 40% of cases.429 Microdissected Hodgkin cells may demonstrate evidence of immunoglobulin heavy-chain gene rearrangements,430 but results of these studies are generally negative on whole bone marrow specimens. Therefore, unless non-Hodgkin’s lym-phoma is in the differential diagnosis, flow cytometry immunophenotyping and gene rearrangement studies are not helpful in the evaluation of a bone marrow specimen for Hodgkin’s disease.

Non-neoplastic Lymphoid Aggregate

The identification of lymphoid aggregates in the bone marrow often causes diagnostic problems, particularly in patients with a history of malignant lymphoma. Because discordance in morphologic features may occur between the original lymphoma and the bone marrow disease, these aggregates cannot be ignored simply because they are morphologically different from the original disease. Non-neoplastic lym-phoid aggregates are common in elderly patients and are more common on clot biopsy material.7,431-433 These non-neoplastic aggregates are usually small and uniformly round without irregular infiltration into the surrounding marrow (Fig. 43-34). They are often associated with small capillar-ies that may be identified within the aggregate. The lym-phoid cells are usually small, but a few admixed large cells and histiocytes may be present. Most lymphocytes in a non-neoplastic or reactive lymphoid aggregate are T cells that immunoreact with CD3, CD43 and bcl-2, but not with CD20 or CD79a. Germinal center formation with predomi-nantly B cells may also occur and is usually a clue to a reactive or autoimmune process.433-435

The neoplastic lymphoid aggregate is often monotonous and irregular in appearance and may have irregular infiltra-tion into the surrounding marrow. Aggregates of small lym-phocytic lymphoma and follicular lymphoma, however, often show a heterogeneous mixture of small cells with admixed larger cells. As mentioned earlier, a paratrabecular aggregate without identifiable bone marrow or fat between the aggregate and the bone in a patient with a history of follicular lymphoma should be considered evidence of bone marrow involvement by lymphoma. In other situations, immunohistochemical or molecular genetic studies may be useful. The neoplastic aggregates of B-cell lymphoma char-

acteristically demonstrate a marked increased in CD20+ B cells.7,436 Aberrant coexpression of CD5 or CD43 in this B-cell population is usually sufficient for a diagnosis of involvement by lymphoma. Coexpression requires an increase in B cells and virtually every lymphoid cell should immunoreact with CD5 or CD43. Although an increased number of BCL2-positive lymphoid cells are present in most neoplastic aggregates compared with non-neoplastic aggre-gates, non-neoplastic T lymphocytes and mantle B lympho-cytes are BCL2 positive. Therefore, BCL2 staining should not be used alone to distinguish neoplastic from reactive proliferations.437 The molecular detection of a gene rear-rangement or a translocation known to be associated with a type of lymphoma may also be useful in characterizing an atypical lymphoid aggregate,414 but these studies should be correlated with the morphologic and immunophenotypic findings. In some cases, the nature of the aggregates cannot be defined with certainty and this should be clearly stated in the report. In these instances, I use the term atypical lymphoid aggregates of undetermined (or unknown) signifi-cance. An international workshop to standardize response criteria for non-Hodgkin’s lymphoma proposed a category of indeterminate for involvement by lymphoma for cases in which lymphoid aggregates are increased in number or size without cytologic or architectural atypia.410

Granulomatous and Histiocytic Disorders

Granulomas of Bone Marrow

Although aggregates of histiocytes may be present on aspi-rate smears, granulomas are best identified on bone marrow trephine and clot biopsy material.438-440 Generally, two types of granulomas are considered in the bone marrow. The lipogranuloma is a collection of histiocytes surrounding adipose tissue and is usually not associated with disease. Epithelioid granulomas without associated adipose tissue may have admixed lymphocytes, plasma cells, neutrophils, or eosinophils and may have associated necrosis. Mycobac-

Figure 43-34 n A non-neoplastic lymphoid aggregate in a bone marrow clot section. These aggregates are more common on clot section, are non-paratrabecular when present on biopsy section, are usually well circum-scribed, and often contain admixed histiocytes.

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teria and fungal organisms should be excluded by special stains in all cases with epithelioid granulomas, and fresh bone marrow aspirate material should be submitted for culture in all patients being evaluated for infectious dis-eases. Patients with immunodeficiency syndromes may have abundant atypical mycobacteria even when well-formed granulomas are not present. In such cases, special stains for organisms are indicated when any increase in histiocytes is noted. Although fungi and mycobacteria are the most common infectious causes of bone marrow granulomas, brucellosis, leprosy, and viral infections may also cause bone marrow granulomas. Occasional infec-tious granulomas, particularly in Q fever, contain a central clear space surrounded by fibrin that may be mistaken for an incidental lipogranuloma.441 Similar fibrin ring granulo-mas have also been described with cytomegalovirus infection.442

Noninfectious granulomas, including those of sarcoid-osis, drug reactions, collagen-vascular disease, and associ-ated with neoplasms, cannot be reliably distinguished from infectious granulomas and appropriate histochemical stains for organisms are indicated in these patients. Bone marrow granulomas are common in patients with Hodgkin’s disease and non-Hodgkin’s lymphomas and may be present in the absence of bone marrow involvement by the neoplasm.443

Hemophagocytic Syndromes

Hemophagocytosis may be related to primary or secondary syndromes and is discussed in more detail in Chapter 42. Primary hemophagocytic lymphohistiocytosis is a rare, fatal childhood disorder that may be familial or sporadic.444 It is usually elicited by a viral infection. Bone marrow involvement may be evident in only 40% of cases that have extensive disease elsewhere.445 Secondary hemophagocyto-sis may be infectious or related to neoplasia.446-448 All types frequently involve multiple organs, including the bone marrow, and have similar morphologic features (Fig. 43-35). Hemophagocytosis consists most prominently of eryth-rophagocytosis that may be best visualized on aspirate smears or imprint slides. The histiocytes have characteristi-

cally bland nuclear features without mitoses or atypia. Hemophagocytosis in the bone marrow may be associated with T-cell malignant diseases, even in the absence of marrow involvement by lymphoma. When present, neo-plastic T cells are usually distinct from the reactive-appear-ing histiocytes that are engulfing other hematopoietic cells, which may include phagocytosis of neoplastic cells. Other bone marrow elements, particularly granulocytes and ery-throid precursors, may be relatively decreased in number. Lymphoid cells, including immunoblasts, may be increased in the bone marrow in association with hemophagocytosis. The presence of immunoblasts does not necessarily indicate the presence of an associated lymphoma. Cytologic atypia of the lymphoid cells should be easily identified in lym-phoma-associated hemophagocytosis. Gene rearrangement studies may be helpful in making a primary diagnosis of lymphoma on a bone marrow specimen that has extensive hemophagocytosis. In situ hybridization for EBV may also be useful because hemophagocytosis may be secondary to EBV infection or to an EBV-related NK or T-cell malignant disease.

Storage Diseases

Various storage diseases may involve the bone marrow, but these diseases are generally rare and should be diagnosed and classified by identification of the enzymatic defect char-acteristic for each disease.449 Gaucher’s disease and Niemann-Pick disease are the most common storage diseases encountered in the bone marrow and both may cause the accumulation of patchy or diffuse aggregates of large histio-cytes with abundant cytoplasm and small nuclei.450 On smears, Gaucher cells have slightly basophilic cytoplasm that is often compared to crumpled tissue paper, in contrast to the finely vacuolated cytoplasm of the characteristic cells of Niemann-Pick disease. Small histiocytes with more baso-philic cytoplasm and vacuoles are often termed sea-blue histiocytes and are also seen in Niemann-Pick disease. None of these cell types is specific for a given disease and sea-blue histiocytes may also be seen in association with lipid disor-ders, infectious diseases, red blood cell disorders, and myeloproliferative neoplasms.

Figure 43-35 n Hemophagocytic syndrome. A, Large histiocytes engulfing red blood cells are identified on touch preparations or aspirate smears. B, Sheets of histiocytes may be present in the bone marrow biopsy.

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Nonleukemic Histiocytic Disorders

Histiocytic disorders, other than acute and chronic leuke-mias, are rare in the bone marrow. Langerhans’ histiocytosis, sinus histiocytosis with massive lymphadenopathy (SHML), and true histiocytic lymphoma generally present in other organs and only rarely and focally involve the marrow.

So-called malignant histiocytosis or true histiocytic lym-phoma involving the bone marrow is controversial. Some cases of these diseases seem to represent extramedullary presentations of acute monoblastic leukemia without obvious peripheral blood involvement, similar to other extramedullary myeloid tumors, and minimal bone marrow involvement.451,452 Previously, cases of hemophagocytic syn-drome (with or without associated malignancy), anaplastic large cell lymphoma, and other malignant lymphomas with bone marrow involvement were interpreted as malignant histiocytosis.453,454 Because of the historic heterogeneity of diseases covered by this term, as well as the current lack of specific features that distinguish malignant histiocytosis of the bone marrow from monoblastic leukemia, I do not use this term in the diagnosis of primary bone marrow proliferations.

Mast Cell Disease

Generalized mast cell disease, or mastocytosis, has been subdivided into systemic mast cell disease, which is usually associated with skin lesions and a good prognosis, and malignant mast cell disease, which does not characteristically involve the skin, is often associated with other hematologic malignant diseases, and has a generally poor prognosis.455,456 The WHO classification considers both these disorders as systemic mastocytosis with specific categories of indolent systemic mastocytosis, systemic mastocytosis with associ-ated clonal hematologic non–mast cell lineage disease, aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma.42 All these types may involve the bone marrow,457,458 and it is not possible to differentiate these disorders on a bone marrow biopsy alone, with the excep-tion of detecting the presence of a clonal hematologic non–mast cell lineage disease.

On aspirate smear material, mast cells are usually admixed with hematopoietic cells within the marrow parti-cle and an increase in the cells may be overlooked. Mast cells are most prominent within the cellular particles of smears, and abnormal mast cells are usually spindled in appearance and may show loss of granules. Abnormalities of other marrow cells may be best identified on the smears. The most common histologic pattern of bone marrow involvement is one of patchy paratrabecular or perivascular stellate aggregates of fibrosis with admixed mast cells, lym-phocytes, and eosinophils (Fig. 43-36).459 The mast cells are easily overlooked in these aggregates and may have a spindle appearance similar to fibroblasts or histiocytes, or they may represent small round cells with slightly granular, baso-philic cytoplasm that may be mistaken for lymphocytes or plasma cells. This perivascular and paratrabecular pattern of mast cells may also be associated with surrounding marrow changes of AML, myelodysplasia, or a chronic myeloproliferative neoplasm (often unclassifiable).460-462

Sheets of atypical mast cells may rarely be evident on bone marrow sections. This pattern is more commonly associated with peripheral blood involvement by mast cells (mast cell leukemia). Osteosclerosis may be seen with any pattern of marrow involvement.

On tissue sections, mast cells usually test positive for Giemsa, toluidine blue, and chloracetate esterase stains, but some of these cytochemical reactions are pH dependent in neoplastic mast cells.463 Immunohistochemical studies show the mast cells to test positive for CD43, CD68, CD117, and tryptase, with tryptase the most specific marker.464 Neoplas-tic mast cells also tend to show aberrant expression of CD2 and CD25.459,465 Because of the patchy, focal nature of the bone marrow disease, some cases may only show a slight interstitial increase in mast cells on immunohistochemical analysis without obvious aggregates. In the proper clinical setting, in which the patient has clinical features of mast cell disease and an elevated serum total tryptase level, the detection of an interstitial increase in mast cells is consis-tent with, but not diagnostic of, mastocytosis.466,467

Except for cases of mast cell leukemia, the peripheral blood changes of mast cell disease are not specific.462 In systemic mast cell disease, the peripheral blood is often normal, whereas cytopenias are common in the presence of an associated malignant disease.

Metastatic Tumors

Metastatic tumors in the bone marrow may be identified in staging procedures or may be unexpected findings in a patient undergoing an evaluation for abnormal blood counts.468 A bone marrow biopsy may be a relatively easy way to identify and classify a pediatric tumor without resec-tion of the primary mass. The most common nonhemato-logic malignant diseases to involve the bone marrow are the following: prostate, breast, and lung carcinomas; neuroblas-toma; Ewing’s sarcoma/peripheral neuroectodermal tumor (PNET); rhabdomyosarcoma; and malignant melanoma.469,470 Tumor is often not easily identifiable on aspirate smears, but when present it is more often seen on the feathered

Figure 43-36 n Mast cell disease in a patient with myelodysplasia. The focus of marrow fibrosis contains mast cells admixed with eosinophils. The surrounding bone marrow is hypercellular and myelodysplastic changes were evident on the bone marrow aspirate smears.

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edges of the smears as small clumps of cells (Fig. 43-37). When the primary tumor type is unknown, immunohisto-chemical studies may be useful to identify the tumor type. In adults, most metastatic tumors are carcinomas or malig-nant melanoma, although sarcomas may rarely involve the bone marrow. Immunohistochemical studies for keratin and S-100 are useful as initial screens to identify keratin-positive carcinoma and keratin-negative, S-100–positive malignant melanoma. As indicated, additional immunohistochemical studies may be useful to identify estrogen-receptor–positive breast or gynecologic carcinomas, prostate-specific antigen–positive prostate tumors, thyroid transcription factor 1 (TTF1)–positive thyroid or lung carcinomas, and chromo-granin-positive small cell carcinomas. Keratin subset analy-sis, as in other sites, can also be useful in identifying the primary site of a carcinoma metastatic to the bone marrow.471-473

Pediatric sarcomas in the bone marrow display a similar immunohistochemical staining profile as seen in other sites, and the primary diagnosis can be made on a bone marrow biopsy specimen in many cases. The small blue round cell tumors may be confused with acute leukemic infiltrates of the bone marrow. An initial immunohistochemical panel that includes vimentin, keratin, desmin, CD99, myeloper-oxidase, and TdT is often helpful in the evaluation of these tumors. Although CD99 is characteristic of Ewing’s sarcoma/PNET, ALL specimens also test positive for this marker. TdT staining is helpful in this differential diagnosis and is posi-tive in ALL but negative in Ewing’s/PNET. Lack of vimentin is characteristic of neuroblastoma. Details of the morpho-logic, immunohistochemical, and molecular genetic fea-tures of these tumors are described in other chapters, including Chapters 7 and 46.

The routine use of immunohistochemical studies is usually not necessary in the evaluation of staging or post-therapy bone marrows of patients with the known disease. Metastatic tumor cells are usually large, occur in aggregates, and have associated fibrosis with or without necrosis in the marrow. When foci of fibrosis are present without obvious tumor, selected immunohistochemical studies may be useful to further exclude tumor cells within the fibrous stroma. Lobular carcinoma of the breast, however, often infiltrates the bone marrow as individual

small cells without associated fibrosis and may be easily overlooked on H&E-stained sections alone.474,475 Because the detection of these individual cells within the marrow appears to have prognostic significance, routine staining for keratin on bone marrow biopsy and clot sections from patients with lobular carcinoma should be considered. The identification of keratin-positive cells by immunohisto-chemistry allows for the repeat review of the suspicious area on the routine section and usually identification of indi-vidual tumor cells on those sections.

Peripheral blood involvement by metastatic tumor, termed carcinocythemia, is extremely uncommon and usually represents a late event with short survival.476 Other abnormalities of the blood in patients with metastatic car-cinoma are anemia with teardrop-shaped red blood cells, leukoerythroblastosis, leukocytosis, and microangiopathic hemolytic anemia.

APPROACH TO THE NON-NEOPLASTIC BONE MARROW

Aplasias

A decrease in bone marrow cellularity for a patient’s age may be related to a variety of causes. Artifactual “hypocellular-ity” resulting from sampling of only subcortical marrow should not be misinterpreted as aplasia and an accurate estimate of marrow cellularity cannot be made on very small biopsy specimens that contain only the subcortical marrow space. True hypocellularity may result from a decrease in all bone marrow cell lines or only selected cell line decreases.

Aplastic Anemia

Aplastic anemia may be acquired or congenital and repre-sents a decrease in granulocytic, erythroid, and megakaryo-cytic cells.477,478 Criteria for severe aplastic anemia require (1) bone marrow of less than 25% cellularity or less than 50% cellularity with less than 30% hematopoiesis and (2)

Figure 43-37 n A, Neuroblastoma on a bone marrow aspirate with rosette formation. B, Clumps of metastatic lobular breast carcinoma are identified at the feathered edge of a bone marrow aspirate smear.

A B

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two of the following three peripheral blood findings: a neu-trophil count of less than 0.5 × 109/L, a platelet count of less than 20,000/µL, or anemia with an absolute reticulo-cyte count of less than 4 × 109/L. Chronic moderate aplastic anemia can be diagnosed in patients who do not meet cri-teria for severe aplastic anemia but who have moderately depressed peripheral blood counts for more than 3 months. The peripheral blood of aplastic anemia demonstrates pan-cytopenia without obvious abnormalities of the circulating cells.479 Aspirate smears usually show small, paucicellular particles containing histiocytes, mast cells, lymphocytes, and plasma cells with no or very rare normal hematopoietic cells. The biopsy specimens in these patients are variably hypocellular. Some have only rare hematopoietic cells, rep-resenting less than 5% of the marrow cellularity, whereas others have large areas of markedly hypocellular marrow admixed with more normocellular areas. Interstitial small lymphocytes and plasma cells, as well as well-formed lym-phoid aggregates, are common. Aplastic bone marrow may be seen after toxic exposure, including chemotherapy or radiation therapy, or viral infection, including parvovirus and hepatitis, and with pregnancy, all of which may result in only transient aplasia. The post-therapy biopsy often dem-onstrates transient marrow aplasia with marrow edema and eosinophilic proteinaceous fluid accumulation in the bone marrow.

Other diseases may mimic aplastic anemia. Hypocellular acute leukemias and MDSs have a similar appearance and may result in inaspirable material for evaluation,480,481 and a subset of patients with apparent aplastic anemia will develop myelodysplasia or acute leukemia.482 The identifi-cation of an increase in CD34+ immature cells in the bone marrow (>5%) is helpful in the identification of these dis-eases. Cytogenetic studies are also useful in this differential diagnosis to identify cytogenetic abnormalities commonly associated with MDSs. Patients with Fanconi’s anemia may develop marrow aplasia and may also develop myelodyspla-sia and acute leukemia.483 HCL may mimic aplastic anemia, but the identification of increased numbers of interstitial B lymphocytes in the bone marrow of HCL excludes the diag-nosis of aplastic anemia.

Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) may cause pan-cytopenia and marrow aplasia similar to aplastic anemia or may result in erythroid hyperplasia.484 PNH is characterized by recurrent episodes of hematuria that are secondary to intravascular hemolysis. It is a stem cell disorder that is often related to somatic mutation of the X-linked PIGA gene.485 This mutation results in a deficiency of the glyco-sylphosphatidylinositol (GPI)–anchoring proteins of decay-accelerating factor (DAF or CD55) and the membrane inhibitor of reactive lysis (MIRL or CD59) on red blood cells, granulocytes, monocytes, and platelets. Loss of these proteins results in the increased sensitivity of the red blood cells to the lytic action of complement. The antigen loss may be detected by flow cytometry evaluation of multiple cell lines.486 PNH is characterized by the presence of chronic hemolytic anemia, and although positive sucrose hemolysis and Ham tests were used in the past for diagnosis, flow cytometry immunophenotyping of peripheral blood is the

current diagnostic method of choice. That the defects of PNH may also be present in patients with acute leukemia, myelodysplasia, and aplastic anemia suggests an association of PNH with these diseases.487

Serous Fat Atrophy

Serous degeneration, also known as serous fat atrophy, is associated with hypocellular bone marrow and represents mucinous or gelatinous degeneration of the fatty marrow elements (Fig. 43-38).488,489 On aspirate, pink to blue stain-ing material may be seen surrounding fat cells. On the biopsy material, the fat cells are decreased in size and are surrounded by amorphous pink material. Such degenera-tion in the bone marrow represents a systemic change in the total body adipose tissue. Serous fat atrophy is seen with a variety of diseases that are associated with malnutrition and emaciation, including anorexia nervosa, chronic renal disease, malignant diseases, acquired immunodeficiency syndrome, and tuberculosis.

Red Cell Aplasias

Aplasia of a single cell line in the bone marrow is less common than are the trilineage aplasias described earlier, and it may be congenital or acquired. Diamond-Blackfan anemia is an autosomal recessive cause of pure red cell aplasia that is often associated with other abnormalities; the anemia becomes apparent shortly after birth with persistent elevations of hemoglobin F, i antigen, and red blood cell macrocytosis.490 Other causes of red cell aplasia are drug or toxin exposure, infections, and thymoma or CLL.491 Parvo-virus infection causes erythema infectiosum (fifth disease) in children and infects red blood cell precursors, which may result in a red cell aplasia.492,493 The virus causes the few residual erythroblasts to enlarge, with large intranuclear inclusions (Fig. 43-39). All causes of red cell aplasia have similar morphologic features, with the exception of the characteristic nuclear inclusions of parvovirus infection. All demonstrate a marked decrease in red blood cell precursors with only rare scattered erythroblasts present. Transient red blood cell aplasia (transient erythroblastopenia of childhood) has been described in young children that is not known to be associated with any viral or toxic insult.494,495

Figure 43-38 n Serous fat atrophy in a bone marrow biopsy.

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Granulocyte Aplasia

Congenital causes of granulocyte aplasia include Shwach-man-Diamond syndrome, Kostmann’s syndrome, and cyclic neutropenia.496,497 Acquired granulocytopenia may occur secondary to drugs, infections, hypersplenism, and autoim-mune disease. Acquired granulocytopenias occur secondary to peripheral destruction or redistribution of granulocytes, and bone marrow examination of these patients may not demonstrate a decrease in granulocytic cells.498 Although parvovirus infection is usually associated with red cell aplasia, it may also cause chronic neutropenia.499 The con-genital causes of granulocyte aplasia result in a decrease in bone marrow granulocytes that are usually normal in appearance; however, maturation arrest at the promyelocyte and myelocyte stages is seen in Kostmann’s syndrome.497

Megakaryocyte Aplasia

Megakaryocyte aplasia is uncommon and most cases of megakaryocyte-poor thrombocytopenia probably represent an unusual form of myelodysplasia primarily involving that cell lineage.500,501 In newborns, megakaryocyte hypoplasia is associated with the thrombocytopenia with absent radius (TAR) syndrome.502 Amegakaryocytic thrombocytopenia has also been reported as a result of parvovirus B19 infection.503

Hyperplasias

Non-neoplastic hyperplasias of one or more bone marrow cell lines are often related to changes outside the bone marrow and must be correlated with peripheral blood find-ings, other appropriate laboratory data, and clinical infor-mation. Patients recovering from toxic insults, including chemotherapy and radiation therapy, demonstrate trilineage bone marrow hyperplasia. In a similar fashion, destruction of a particular cell line outside of the marrow, as is seen in some autoimmune diseases, results in hyperplasia of the corresponding cell line in the marrow. Because the bone marrow changes may represent a reaction to events

elsewhere in the body, the bone marrow specimen alone is often not diagnostic of the patient’s underlying disease process.

Erythroid Hyperplasia

Erythroid hyperplasias represent a response to peripheral red blood cell loss or destruction or are related to the inef-fective production of normal red blood cells, as seen in hemoglobinopathies. The causes of erythroid hyperplasia are best addressed in combination with peripheral blood features of the red blood cells.504-506 In general, ancillary laboratory testing is needed to characterize the cause of the erythroid hyperplasia precisely. Dyserythropoiesis, particu-larly mild irregularities of the nuclear contours of erythroid precursors, is common in cases of florid erythroid hyper-plasia and should not be overinterpreted as evidence of myelodysplasia.

erythroid hyperplasia associated with normocytic anemia

Hemorrhage, hemolytic anemia, intrinsic bone marrow disease (including aplastic anemia or malignancies), and anemia of chronic disease are the most common causes of erythroid hyperplasia associated with normocytic anemia in patients with no history of a toxic insult, chemotherapy, or hemoglobinopathy. Following hemorrhage or with hemo-lytic anemia, the peripheral blood has an increase in poly-chromatophilic red blood cells resulting from reticulocytosis. Hemolytic anemias may be characterized by microsphero-cytes and fragmented red blood cells in the peripheral blood. The bone marrow changes of the various causes of normocytic anemia with erythroid hyperplasia are similar, with an increase in red blood cell precursors. This increase is usually accompanied by a left shift in erythroid precur-sors with increased numbers of pronormoblasts. Such a left shift should not be interpreted as megaloblastic change, which represents an enlargement of both myeloid and ery-throid precursors. Iron staining of a bone marrow aspirate smear and laboratory iron studies are appropriate in nor-mocytic anemias of unknown cause because the early stages of iron deficiency may manifest as normocytic anemia with erythroid hyperplasia. Combined anemias, such as vitamin B12 or folate deficiency coupled with iron deficiency or thalassemia, may also manifest with normocytic red blood cell indices.

erythroid hyperplasia associated with macrocytic anemia

Erythroid hyperplasia associated with macrocytic anemia may be related to megaloblastic anemia or to other causes. Mega-loblastic anemias are the result of deficiencies of vitamin B12 or folate and demonstrate peripheral blood changes that include macrocytic anemia and hypersegmentation of neu-trophils.507,508 Bone marrow examination is not usually per-formed for this diagnosis. The bone marrow aspirate characteristically shows a left shift of erythroid cells with enlarged pronormoblasts and normoblasts with fine nuclear chromatin that may be mistaken for myeloblasts. The ery-throid series in the marrow also demonstrates multinucle-ation and nuclear-to-cytoplasmic asynchrony. The maturing granulocyte series is also abnormal in megaloblastic anemia with enlarged or “giant” metamyelocytes and band neutro-phils present. Megakaryocytes may show hypersegmenta-

Figure 43-39 n Bone marrow biopsy from a child with parvovirus infec-tion. The erythroid precursors are enlarged, with nuclear clearing.

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tion of nuclei. These features should be correlated with vitamin B12 and folate levels and may be confused with changes of myelodysplasia or acute leukemia. Cases of severe hemolytic anemia or hemorrhage result in macro-cytic anemia with a high reticulocyte count and marked left shift of erythroid precursors of the marrow. Congenital dys-erythropoietic anemia (CDA) results in an erythroid hyper-plasia, and types I and III are associated with a macrocytic anemia (type II may be normocytic).509 CDA is associated with abnormal bone marrow erythroid cells with intranu-clear bridging and varying degrees of multinucleated ery-throid cells. The most bizarre multinucleated cells are present in type III, more than one-third binucleate cells in type II, and intranuclear chromatin bridges and less than one-third binucleate cells in type I. Type II is also termed HEMPAS (hereditary erythroblastic multinuclearity with a positive acidified serum test), which differs from PNH by a negative sucrose hemolysis test result. Other disorders resulting in erythroid hyperplasia with macrocytic anemia include alcohol ingestion, liver disease, cytotoxic drugs, hypothyroidism, pulmonary disease, aplastic anemia, and myelodysplasia,508,510 and these disorders may not have associated marrow hyperplasia.

erythroid hyperplasia associated with microcytic anemia

Iron deficiency is the most common cause of erythroid hyperplasia associated with microcytic anemia.511,512 Patients with iron deficiency develop microcytic anemia with hypo-chromic red blood cells and prominent anisopoikilocytosis with elliptical or elongated red blood cells. Thrombocytosis is also often present. Iron staining of bone marrow aspirate material and serum iron or ferritin studies are usually diag-nostic for iron deficiency. Lead toxicity may also result in a similar peripheral blood and bone marrow pattern and may be accompanied by iron deficiency.513 The presence of coarse basophilic stippling of peripheral blood red cells and bone marrow erythroid cells is seen in lead toxicity and not in the usual case of iron deficiency. Thalassemias and hemo-globin E disease and trait also result in microcytic anemia, although the total red blood cell count of the peripheral blood is often normal in thalassemia in comparison with the decrease in red blood cells of iron deficiency.511 Some anemias of chronic disease and hereditary sideroblastic anemias also result in microcytic anemia. Despite the cause, all demonstrate erythroid hyperplasia in the marrow and cannot be reliable distinguished without iron studies and other appropriate laboratory tests.

White Blood Cell Hyperplasiasneutrophilia

Peripheral blood neutrophilia is usually not associated with bone marrow granulocytic hyperplasia and results from demargination of neutrophils or a shift of neutrophils to the blood from other organs. Bone marrow granulocytic hyper-plasia usually occurs in response to an infection, allergic reaction (usually with eosinophilia), toxic events, drug reaction, or association with neoplasia. The ratio of granu-locytes to erythroid precursors may increase with a left shift of myeloid cells. In infectious disease, the peripheral blood neutrophils often demonstrate toxic granulation and Döhle

bodies. In neoplastic conditions, this granulocytic hyper-plasia of the marrow may not be associated with marrow involvement by the neoplasm.514 Although organisms, including parasites, may rarely be identifiable on the bone marrow smears or biopsy, the cause of the granulocytic hyperplasia is usually not apparent. The primary differential diagnosis of non-neoplastic granulocytic hyperplasia is CML or another myeloproliferative neoplasm. The lack of basophilia or clusters of atypical megakaryocytes and the results of cytogenetic studies for t(9;22) are helpful in excluding the diagnosis of CML.

monocytosis

Reactive monocytosis is a nonspecific finding and may be seen with infections, sarcoidosis, collagen-vascular diseases, hematologic disorders, and malignant diseases.515,516 The hematologic disorders include hemolytic anemia, and reac-tive monocytes may be increased in the peripheral blood or bone marrow in patients with Hodgkin’s disease, non- Hodgkin’s lymphoma, and carcinomas. Neoplastic mono-cytes in the peripheral blood with acute myelomonocytic leukemia and CMML may have features similar to reactive monocytes, and bone marrow examination is necessary to confirm and classify the leukemic process in those patients.

eosinophilia

The most common cause of peripheral blood or bone marrow eosinophilia worldwide is parasitic infections, although drugs and allergic reactions are the most common causes in developed countries.517 Other causes of eosino-philia include asthma, inflammatory bowel disease, vascu-litides, and malignant diseases. Reactive eosinophilia may be prominent in patients with colon carcinoma, Hodg-kin’s disease, T-cell lymphomas, and myeloproliferative neoplasms. Persistent eosinophilia without an identified underlying cause should raise concern for chronic eosino-philic leukemia or one of the related myeloproliferative neoplasms.

basophilia and mast cell hyperplasia

Non-neoplastic peripheral blood and bone marrow baso-philia is usually mild and is commonly overshadowed by more noticeable eosinophilia. This basophilia is most com-monly associated with allergic or inflammatory conditions, including asthma, allergic rhinitis, nasal polyposis, and atopic dermatitis.518 Significant increases in basophils, often abnormal, are seen in the peripheral blood and bone marrow of patients with CML and acute leukemias that are associ-ated with t(9;22) and t(6;9). Elevations of non-neoplastic bone marrow mast cells are common in association with chronic lymphoproliferative disorders in the bone marrow. These mast cells are evenly dispersed throughout the marrow particles and do not form the aggregates and foci of fibrosis that are seen with bone marrow involvement by mast cell disease.

lymphocytosis

Reactive lymphocytosis is most commonly related to viral infections, particularly infectious mononucleosis and viral hepatitis, and it may also be seen with bacterial infections. The reactive cells are usually CD8+ T cells and are large with abundant cytoplasm. In children, reactive lymphocytosis C


with cleaved and irregular T-lymphocyte nuclei may be seen in Bordetella pertussis infection.519 Transient T-cell lympho-cytosis may also result from trauma, acute cardiac disease, and epinephrine administration.520,521 B-cell lymphocytosis is less common. Persistent polyclonal B lymphocytosis occurs most frequently in young and middle-aged women and is associated with smoking, elevated serum IgM, and the pres-ence of the HLA-DR7 antigen.522,523 Patients have persistent lymphocytosis, usually between 4 and 14 × 109/L, with binucleated peripheral blood lymphocytes. Increased lym-phocytes, including bilobed lymphocytes (Fig. 43-40), are present in the bone marrow and nodular lymphoid aggre-gates may be present. The cells are polyclonal CD19+/CD20+/CD22+ B cells that do not usually express CD5 but are FMC7 positive. The chromosomal abnormality i(3q) is reported in a majority of patients, but the clinical course is benign. Immunoglobulin heavy-chain gene rearrangements are not detected in these cases. Rare cases of CD5+ reactive lymphocytosis have also been described.524

Megakaryocytic Hyperplasias

Non-neoplastic megakaryocytic hyperplasias are associated with either thrombocytopenia or thrombocytosis. Throm-

bocytopenia-associated megakaryocytic hyperplasia is usually caused by an autoimmune disorder, particularly idiopathic (or immune) thrombocytopenic purpura. The bone marrow in these cases shows an increased number of normal appearing megakaryocytes and small, more imma-ture megakaryocytes.

Non-neoplastic megakaryocytic hyperplasia associated with thrombocytosis may occur in chronic infections, the recovery phase of acute infections, collagen-vascular disease, hemolytic anemias, iron deficiency anemia, and malignant disease. Rebound thrombocytosis may occur after recovery from thrombocytopenia and following splenectomy. Reactive thrombocytosis must be differentiated from the myeloproliferative neoplasms, particularly ET. Reactive thrombocytosis rarely demonstrates a platelet count as high as 1000 × 109L, and the atypical megakaryocytes and the marked degree of megakaryocytic hyperplasia of the myelo-proliferative neoplasms are usually not present in reactive conditions.525

Hyperplasias Related to Growth Factors

Bone marrow hyperplasia related to growth factor admi-nistration is most often related to erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), and granu-locyte-macrophage colony-stimulating factor (GM-CSF).526 Administration of growth factor should always be docu-mented in the clinical information for bone marrow speci-mens. The erythroid hyperplasia caused by EPO does not usually cause diagnostic dilemmas. However, some diffi-culty may be encountered when changes of G-CSF or GM-CSF are not recognized. These factors usually result in peripheral blood neutrophilia, but leukoerythroblastosis, monocytosis, or eosinophilia may occur. Toxic granulation of neutrophils with Döhle bodies may mimic infectious leukocytosis. The bone marrow may demonstrate granulo-cyte hyperplasia with left shift that may range from hypocel-lular bone marrow with aggregates of left-shifted immature cells, mimicking hypocellular acute leukemia, to hypercel-lular bone marrow with granulocytic hyperplasia mimick-ing a myeloproliferative neoplasm, to maturation arrest (Fig. 43-41). Although granulocyte maturation occurs over

Figure 43-40 n A binucleated peripheral blood lymphocyte in a woman with polyclonal B-cell lymphocytosis.

Figure 43-41 n Growth factor administration may cause marked granulocyte predominance and left shift on the bone marrow aspirate smear (A) and biopsy (B) that may be mistaken for residual acute leukemia or a myeloproliferative neoplasm if not suspected.

A B

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time following growth factor administration, a single bone marrow specimen may demonstrate a predominance of immature myeloid cells, usually at the promyelocyte and myelocyte stage of differentiation. These findings may suggest a diagnosis of de novo acute promyelocytic leuke-mia or of relapsed AML. The promyelocytes in these speci-mens are usually large with a prominent perinuclear hof, do not contain Auer rods, and are not associated with increased numbers of myeloblasts. A predominance of such cells in a specimen should raise the suspicion of G-CSF or GM-CSF therapy. Even when a history of growth factor therapy is known in a patient with a history of AML, the differential diagnosis between relapse and growth factor effect may be difficult. In such cases, immunopheno-typing studies to identify an aberrant phenotype character-istic of the patient’s original leukemia may be helpful, as may cytogenetic studies to identify a leukemic clone. Alter-natively, a repeat aspirate specimen may be obtained several days after stopping the growth factor to see whether the immature cell population has disappeared (loss of growth factor affect) or increased (relapsed leukemia affect). Patients receiving megakaryocytic growth factors are less likely to undergo bone marrow biopsy after therapy, but these agents result in a proliferation of megakaryocyte precursors.527

Other Non-neoplastic Marrow Changes

Fibrosis

Fibrosis of the bone marrow may occur in association with a variety of neoplastic and non-neoplastic conditions, but it is a nonclonal proliferation of fibroblasts.528 Fibrosis is usually of the reticulin type, as detected by silver stains, in the early stages, but may progress to a collagen fibrosis that is detectable on trichrome stains. Reticulin fibrosis of the bone marrow is normally absent or minimal, although it increases slightly with age.529 Extensive marrow fibrosis typically causes erythroblastosis of the peripheral blood that is characterized by a left shift of granulocytes, enlarged platelets, teardrop-shaped red blood cells, and nucleated red blood cells. Diffuse fibrosis usually results in the inability to aspirate marrow particles. Bone marrow fibrosis is common in myeloproliferative neoplasms, particularly PM. Many other neoplasms involving the marrow, including some acute leukemias, malignant lymphomas, and meta-static tumors, result in focal or diffuse marrow fibrosis (Table 43-19). Bone marrow fibrosis may also be associated with non-neoplastic conditions, especially inflammatory or reparative changes and metabolic disorders. Renal osteodys-trophy and primary hyperparathyroidism cause extensive marrow fibrosis with a relative decrease in normal bone marrow elements (Fig. 43-42). Extensive bone remodeling with abundant osteoblasts is also common and multinucle-ated giant cells may be present. Because bone marrow fibro-sis is a reactive proliferation of fibroblasts triggered by growth factor production of neoplastic cells or hormonal changes related to metabolic disease, treatment of the underlying disease process usually results in resolution of the fibrosis.

Necrosis

Bone marrow necrosis is an expected finding following chemotherapy for hematopoietic tumors, and early post-therapy bone marrow specimens may be acellular with only fibrinous, eosinophilic necrotic material present. In time, regenerative changes occur admixed with the fibrinous necrosis. Other forms of bone marrow necrosis that are not treatment related are uncommon,530,531 but they are gener-ally assumed to be associated with infarction or related to infectious diseases, such as necrotizing granulomas of tuberculosis. Zonal areas of tumor necrosis with ghosts of tumor cell nuclei may be seen in bone marrow of patients with sickle cell crisis, with hematopoietic malignant dis-eases such as acute leukemia or malignant lymphoma, or adjacent to areas of viable metastatic tumor, even before chemotherapy. On smears, necrotic cells are smudged without clear cell borders and are difficult to classify further. With extensive bone marrow necrosis, patients may have bone pain and fever.

taBle 43-19Conditions Associated with Bone Marrow Fibrosis

Fibrosis Associated with Malignant Diseases*

primary myelofibrosisother myeloproliferative neoplasmsacute megakaryoblastic leukemiaother acute myeloid leukemiasacute lymphoblastic leukemiaHairy cell leukemiamast cell diseasemalignant lymphoma, particularly of follicular center cell originHodgkin’s diseaseCarcinoma

Fibrosis Associated with Non-neoplastic Conditions

Renal osteodystrophyprimary hyperparathyroidismHypoparathyroidismVitamin d deficiency

*Fibrosis may be present even in the absence of marrow involvement by the neoplasm.

Figure 43-42 n Prominent osteoblast proliferation and bone marrow fibrosis in a patient with primary hyperparathyroidism. C


BONE MARROW REPORT

Table 43-20 lists the suggested elements to include in the bone marrow report. Many elements of the report, as well as which tests are performed, are determined by the initial morphologic findings and clinical situation; therefore, the clinical indication for the bone marrow examination should be included. Available complete blood cell count data should also be included as well as review of the peripheral blood smear. The bone marrow aspirate and biopsy exami-nations should describe the three main cell lines of the marrow and, as appropriate, should describe other abnor-mal cellular elements. In the case of infiltrative lesions in the marrow, the cell type and percentage of involvement should be included. Results of all ancillary studies should ideally be included in the final report and correlated with the final diagnosis. This may require amending the report to include molecular genetic or cytogenetic results. This

Abnormalities of Bone Trabeculae

The bone trabeculae should be evaluated in all bone marrow biopsy specimens, even when other abnormalities are appar-ent in the specimen.532 Thinning of the trabeculae is most common, usually resulting from osteoporosis. Thickened trabeculae may be seen in association with metabolic disor-ders or neoplasia. The thickened bone of Paget’s disease is associated with an increase in both osteoclasts and osteo-blasts. The increase in osteoblasts with associated bone resorption and scalloping is seen more commonly in Paget’s disease than in PM, another relatively common cause of thickened bone trabeculae, and marrow fibrosis is usually not a prominent feature of Paget’s disease. Osteosclerotic lesions may also be seen with mast cell disease, multiple myeloma (as part of the POEMS syndrome), metastatic car-cinoma, renal failure, osteopetrosis, and fracture sites, including sites of previous marrow biopsy.

taBle 43-20Checklist of the Contents of the Final Report

Clinical Information

Peripheral Blood Examination

1. Complete blood count data2. Red blood cell changes

a. Red blood cell number and sizeb. Hypochromasia noted, if presentc. description of anisocytosis and poikilocytosis and description of

granules or inclusions, if presentd. increased polychromasia, if present

3. white blood cell changesa. white blood cell numberb. description of cell types with relative hyperplasias, cytopenias, or left

shiftc. description of dysplastic or toxic changesd. presence or absence of blast cells, atypical or neoplastic lymphoid

cells or other abnormal cell populations4. platelet changes

a. platelet numberb. platelet morphology, including granulation and size

Bone Marrow Aspirate Smear/Imprint

1. Red blood cell precursorsa. Relative percentageb. Normal maturation versus left shiftc. dysplastic changes

2. Granulocyte precursorsa. Relative percentageb. Normal maturation versus left shiftc. dysplastic changesd. percentage of blasts and description of blasts if elevated

3. megakaryocytesa. Relative numberb. description of morphology, including size and nuclear features

4. description of other cell types including elevations of lymphocytes, plasma cells, monocytes, mast cells, eosinophils, and basophils

5. differential cell count, if indicated

Bone Marrow Trephine/Clot Biopsy

1. marrow cellularity, including percentage and comparison to normal cellularity for age

2. proportions of myeloid to erythroid cells3. Relative number of megakaryocytes4. Features of bone trabeculae5. degree of fibrosis, if present6. abnormal cellular infiltrates, including granulomas, lymphoid cell

infiltrates, plasma cells, and metastatic tumora. percentage of marrow involvementb. location and pattern (particularly neoplastic lymphoid infiltrates)c. Cell type (lymphoma cell type or differentiation of plasma cell

infiltrate)

Cytochemistry results, If Indicated

1. iron stain results for reticuloendothelial stores and red blood cell incorporation, including the presence and percentage of ringed sideroblasts

2. Cytochemical studies for acute leukemia or hairy cell leukemia, including stains performed and results

3. Reticulin or trichrome stains for fibrosis4. Results of special stains for organisms

Immunophenotyping results, If Indicated

1. method used (e.g., flow cytometry, immunocytochemistry, immunohistochemistry)

2. antibodies/antigens studied3. Cell population studied (e.g., blast cells, lymphoid cells, plasma cells)4. Specimen type studied (peripheral blood, bone marrow aspirate, bone

marrow biopsy)5. Results for each antibody/antigen and interpretation of total findings

Molecular Genetic/Cytogenetic Studies

1. method used (e.g., karyotype analysis, polymerase chain reaction, Southern blot, fluorescence in situ hybridization)

2. test performed (e.g., BCR/aBl1, immunoglobulin heavy-chain gene rearrangements)

3. interpretation of results

Diagnosis

1. tissue examined2. diagnostic interpretation, incorporating all results

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approach allows for a single report that clearly interprets all available data and addresses all findings, including seem-ingly contradictory data that may be easily explained when all the information is considered together.

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