Overview
The hematologic system comprises a variety of different tissue and cell types. These include a diverse range of cell functions and organs, including the bone marrow, spleen, and lymph nodes. Lymph node disease is covered in Chapter 6 . In this chapter, immunohistochemical evaluation of the bone marrow, spleen, and histiocyte/macrophage system will be covered. Where appropriate, immunohistochemical results will be discussed in the context of normal histology. Pathologic processes primary to the site (spleen) or cells types (primary marrow diseases) are discussed.
Bone Marrow Antigens
Markers Commonly Used to Identify Immature Populations
CD34
CD34 is a 115-kDa transmembrane sialomucin encoded on chromosome 1q32.1. Proposed functions include inhibition of differentiation, proliferation, and adhesion. Most of the studies evaluating CD34 expression have used the MY 10 or the QBEND/10 monoclonal antibodies. Staining is described in blasts as membranous with some cytoplasmic staining.
Numerous benign and neoplastic proliferations express this marker, which is not lineage specific and has different meanings in the various contexts. CD34 expression is found on both lymphoid and myeloid blasts and implies immaturity in the context of hematopoietic elements. CD34 expression in nonneoplastic cells may be seen in vascular and rare stromal elements. Neoplastic proliferations with CD34 expression include most vascular tumors, some spindle-cell tumors, and diverse other hematopoietic derived tumors.
CD34 is most frequently used to quantify blasts in bone marrow sections where an aspirate was not obtainable. CD34 is not restricted to the myeloid lineage, as precursor B and T lymphoblasts, hematogones (nonneoplastic early B cell precursors), and blood vessels will also express this marker. In addition, mature megakaryocytes may express CD34 in chronic myeloid stem cell disorders.
CD99
CD99 (p30/32mic2) is a cell surface glycoprotein frequently used to identify peripheral neuroepithelioma and Ewing sarcoma. The monoclonal antibody 013 recognizes a human thymus leukemia antigen and has also been found in immature terminal deoxynucleotidyl transferase (TdT) expressing bone marrow precursors. The staining pattern is cytoplasmic.
As with CD34, CD99 may be used as a marker of immaturity in the bone marrow sections where an aspirate was not obtained. The staining is comparable with TdT; however, it is less sensitive.
CD117
CD117 (C-KIT) is a 145-kDa transmembrane tyrosine kinase receptor that is the product of the KIT -gene located on chromosome 4 (4q11-q12). CD117 is not lineage specific and expression is seen on numerous tissues throughout the body including hematopoietic precursors, mast cells and melanocytes. Staining on myeloid and erythroid precursors and the neoplastic cells of plasma cell myeloma is weak and cytoplasmic. Staining in mast cells is strong and cytoplasmic.
CD117 immunoreactivity has become increasingly important because it is the basis for treatment of gastrointestinal stromal tumors (GIST) with the tyrosine kinase inhibitor imatinib mesylate.
Terminal Deoxynucleotidyl Transferase
TdT is a DNA polymerase that generates antigen receptor diversity by catalyzing the addition of deoxynucleotides to the 3′-hydroxyl terminus of the rearranged immunoglobulin heavy chain and T-cell receptor gene segments. The staining pattern may be nuclear as well as membranous or with paranuclear dot-like positivity.
TdT is generally used in marrow sections to identify and quantify blasts when an aspirate is unobtainable. Caution should be used because hematogones (nonneoplastic early B cell precursors) may also express this marker.
TdT is expressed in the lymphoblasts of acute T or B leukemia/lymphoma, a subset of acute myeloid leukemias (AMLs) and blastic plasmacytoid dendritic cell neoplasm (i.e., hematodermic CD56+/CD4+ neoplasm). Nonhematopoietic malignancies expressing TdT include some pediatric small round blue cell tumors, Merkel cell carcinoma, and small cell lung carcinoma.
Granulocytic/Myeloid Antigens
CD33
CD33 (GP67) is a 67-kDa glycosylated transmembrane protein of the sialic acid binding immunoglobulin-like lectin family that maps to 19q13.1-3 and is thought to play a role in cell adhesion. The staining pattern is membranous.
CD33 is used to identify cells of myelomonocytic derivation and may be helpful to identify suspected myeloid sarcoma in tissue sections. Anti-CD33 targeted chemotherapy (gemtuzumab ozogamicin [Mylotarg]) is used in cases of AML and acute lymphoblastic leukemia (ALL).
CD33 is strongly expressed on myeloid precursors including granulocytes, eosinophils, monocytes, and macrophages/histiocytes. During maturation granulocytes and eosinophils decrease their expression of CD33 while monocytic or myelomonocytic cells do not. CD33 expression is also seen on mast cells and has been reported on one case of plasma cell myeloma. No expression of CD33 is reported on lymphocytes, erythroid precursors, megakaryocytes, or normal plasma cells. Both myeloid blasts and lymphoblasts may express this marker.
Myeloperoxidase
Myeloperoxidase staining recognizes the primary granules in the cytoplasm of granulocytes, eosinophils, and their precursors. The polyclonal antibody staining pattern is cytoplasmic.
Myeloperoxidase is often used as a marker of the myeloid lineage, although cells of monocytic derivation may be weakly positive or nonreactive to the polyclonal myeloperoxidase antibody. Mast cells, plasma cells, lymphoid cells, megakaryocytes, and erythroblasts are typically negative for this marker.
Lysozyme
The lysozyme antibody is polyclonal and is purified from rabbit antiserum after exposure to lysozyme purified from urine from patients with monocytic leukemia. The staining pattern is cytoplasmic.
Lysozyme may be used as a granulocytic marker, although myeloperoxidase has higher specificity. The antibody stains granulocytes and granulocyte precursors, as well as monocytes and macrophages/histiocytes.
Megakaryocytic Markers
CD42b
CD42b (GP Ibα) is a glycoprotein (GP) that is part of the GP Ib-V-IX complex. The staining pattern is cytoplasmic. CD42b is a robust stain in decalcified bone marrow sections with high sensitivity and specificity. Megakaryocytic markers are typically used in bone marrow sections, and rarely other tissues, to identify small immature megakaryoblasts, micromegakaryocytes, or to aid in quantification of mature megakaryocytic forms.
CD61
CD61 (β3 integrin) is a membrane-bound glycoprotein that forms a heterodimer with CD41 (GP IIb) to form the GPIIb/IIIa (CD41/CD61) complex. The staining pattern is cytoplasmic. CD61, similar to CD42b, is used to identify blasts with megakaryocytic differentiation or other megakaryocytes.
Erythroid Markers
CD71
CD71 (transferrin receptor-1) mediates the uptake of transferrin-iron complexes and is highly expressed on the surface of erythroid precursors including erythroblasts. The staining pattern is membranous and cytoplasmic.
CD71 is used to highlight erythroblasts and erythroid precursors with highly sensitive staining results when compared with other erythroid markers such as glycophorin A (CD235a) and hemoglobin. Mature erythrocytes, lymphocytes, and other marrow elements lack significant expression of CD71.
E-cadherin
E-cadherin is mainly expressed by epithelial cells and was thought to be primarily an adhesion molecule. Since its discovery on the developing erythroid cells, a differentiation and signal transduction function has also been proposed. The staining pattern is cytoplasmic.
E-cadherin may be used to identify nonneoplastic erythroblasts and normoblasts. Mature erythroid cells do not express this marker. E-cadherin may be downregulated during erythroleukemia on the developing erythroid cells.
Hemoglobin/Glycophorin A
Hemoglobin is a polyclonal antibody reactive with hemoglobin A and F. The staining pattern is cytoplasmic. Hemoglobin is primarily used to demonstrate the erythroid nature of the blasts of acute erythroleukemia. In addition, hemoglobin may be used to confirm the erythroid nature of the sometimes “worrisome looking” proerythroblasts observed in patients with megaloblastic anemia, myelodysplastic syndromes (MDSs), and chemotherapy-induced megaloblastoid changes. Glycophorin A is a sialoglycoprotein of red blood cell membranes. Immunohistochemical staining has similar qualities to hemoglobin A. Both hemoglobin and glycophorin A are second-line choices for erythroid identification and are less robust stains than either CD71 or E-cadherin.
Other Important Hematopoietic Markers
CD1a
CD1a (R4,T6, CD1, FCB6, HTA1) is a transmembrane glycoprotein that forms a heterodimer with β2 microglobulin to present lipid/glycolipid antigen of self origin, or bacterial origin, to T cells. There are five CD1 genes located on chromosome 1 (1q23.1). CD1a is expressed on indeterminate cells (Langerhans precursors), Langerhans cells, and thymic cortical T cells. The staining pattern is membranous.
CD21
CD21 (complement C3D receptor 2) is a membrane protein on B and T cells that binds complement and acts as a receptor for the Epstein-Barr virus (EBV). The gene is located at 1q32. The staining pattern is membranous.
CD35
CD35 (CR1, C3b/C4b receptor 1 [Knops blood group]) is a membrane glycoprotein found on erythrocytes, leukocytes, glomerular podocytes, and follicular dendritic cells. The function of which is to mediate binding to particles and immune complexes activated by complement. The staining pattern is membranous.
CD25
CD25 recognizes the α chain of the interleukin 2 receptor, and it is thought to be a marker of activation. The staining pattern is cytoplasmic.
CD25 can be used in conjunction with DBA.44 and TRAP in the suspected work up of hairy cell leukemia (HCL) in the bone marrow. The lack of CD25 on otherwise typical cases of HCL may be seen in hairy cell variant cases.
Strong and diffuse expression of tartrate resistant acid phosphatase (TRAP) and DBA.44 are highly sensitive for HCL but may also be seen in extranodal marginal zone lymphoma. T-bet and Annexin-A1 are also immunohistochemical markers used in the suspected work-up of HCL. T-bet has not shown sufficient specificity. Annexin-A1 interpretation is difficult, as the myeloid precursors of the bone marrow will also express this marker. With the recent finding that the majority (over 85%) of hairy cell cases contain the BRAF V600E mutation, immunophenotypic findings may be better supported by molecular testing.
CD25 is also expressed at high levels by activated T cells, regulatory T cells, natural killer (NK) cells, and dendritic cells. An anti-CD25 fused to a diphtheria toxin fusion protein therapy (denileukin diftitox) is used in treatment of some T-cell lymphomas, including some cases of mycosis fungoides.
CD43
CD43 (Leu 22) is a sialomucin expressed on hematopoietic precursors that is thought to play a role in regulation of hematopoiesis. The staining pattern is cytoplasmic.
CD43 may be used as a second line marker of myeloid lineage; however, CD43 is expressed on a variety of normal hematopoietic cells, including normal T cells, most marrow-derived cells, and macrophages/histiocytes. Although not typically coexpressed on “resting” mature B cells, CD43 expression is rarely described on B cells actively producing immunoglobulin. CD43 expression is seen in myeloblasts, immature B cells, lymphoma cells, and rarely in metastases of solid neoplasms. Decreased expression of CD43 on T cells is seen in Wiskott-Aldrich syndrome.
CD68
CD68 (GP110, LAMP4, SCARD1) encoded at 17p13 is a member of the lysosomal/endosomal-associated membrane glycoprotein (LAMP) family of scavenger receptors. CD68 is primarily expressed on lysosomes, endosomes, and neutrophil primary granules with a smaller fraction circulating to the cell surface. The staining pattern is cytoplasmic.
CD68 is often used as a marker of monocytes/macrophages; however, given its organelle specificity, any cell type with an abundance of lysosomes or neutrophil primary granules will express CD68. KP1 and PG-M1 are the antibodies most commonly used to detect CD68; the staining pattern is cytoplasmic. CD68 is expressed throughout the monocytic differentiation pathway, usually more intensely in macrophages than in monocytes. Mast cells may also exhibit CD68 (KP-1) positivity.
CD68 can be used to identify cases of AML with a monocytic component (i.e., formerly AML-M4, M5, especially AML-M5a subtype, which is negative for myeloperoxidase). CD68 is strongly expressed in marrow macrophages in cases of infection associated hemophagocytic syndrome, in T-cell lymphoma with associated histiocytic proliferations, as well as in cases of true histiocytic malignancies. KP-1 is expressed in both cases of systemic mastocytosis and Langerhans cell histiocytosis.
CD123
CD123 (interleukin 3 receptor subunit α) is an interleukin 3 specific subunit of a heterodimeric cytokine receptor. It is frequently expressed on plasmacytoid dendritic cells, where it is thought to provide antiviral resistance and function as a link between the innate and adaptive immunity. The staining pattern is cytoplasmic.
CD123 is used to stain plasmacytoid dendritic cells. It may be used in conjunction with CD2AP, CD56, and BDCA2 for diagnosis of blastic plasmacytoid dendritic cell leukemia. In addition, sheets of CD123 expressing plasmacytoid dendritic cells is a relatively specific finding seen in marrows with chronic myelomonocytic leukemia (CMML). In bone marrow, CD123 may be used in suspected cases of HCL, although it is often weakly expressed or negative in paraffin sections of bone marrow. CD123 is expressed normally by myeloid precursors, macrophages, dendritic cells, mast cells, basophils, and megakaryocytes. CD123 expression may also be seen on blasts of AML.
CD138/Lambda and Kappa Light Chain
CD138, κ, and λ antibodies are discussed at greater length under lymphoid antigens ( Chapter 6 ). They are frequently used together in bone marrow sections to aid in plasma cell quantification and diagnosis of plasma cell disease, as well as following therapy. The staining pattern of CD138 is membranous. The staining pattern of κ and λ are cytoplasmic. Other markers that can identify plasma cells include MUM1 (which may stain activated B cells), CD79a (also stains B cells), CD38, epithelial membrane antigen (EMA) (stain epithelial elements), and CD30 (a small subset of plasma cells).
κ and λ are used primarily to evaluate light chain restriction, although nonspecific background staining may make these antibodies difficult to interpret. In bone marrow samples, κ and λ identify cytoplasmic light chains but not surface light chains. As such, they are of little value in the detection of clonal B lymphocytes, which contain surface light chains but no cytoplasmic immunoglobulin.
κ and λ in situ hybridization is more sensitive and specific and has the advantage of lower background staining; however, it may be more affected by decalcification than immunohistochemical staining.
CD163
CD163 (M130, MM130, SCARI1) is encoded on chromosome 12 (12p13.3). CD163 is a member of the scavenger receptor cysteine-rich super family of proteins whose main function is to scavenge hemoglobin-haptoglobin complexes and thus protect the body from free hemoglobin oxidative damage. It is thought to also function as an innate immune sensor for bacteria and as a means to produce local inflammation. CD163 is expressed on monocytes (typically in the blood) and macrophages (monocytes that have migrated into tissue). CD163 expression is largely confined to cells of the monocytic/macrophage lineage. The staining pattern with the commercially available antibody 10D6 is membranous.
CD207/Langerin
CD207 (Langerin, CLEC4K) is a calcium-dependent (C-type) lectin encoded at 2p13. It is expressed in the Birbeck granules of Langerhans cells, immature dendritic cells of the epidermis, and mucosa. CD207 is thought to function as a means for the Birbeck granule to internalize antigen and start the nonclassical antigen processing pathway. CD207 is expressed on normal Langerhans cells as well as on Langerhans cell histiocytosis, separating this entity from the non-Langerhans cell histiocytic proliferations. This antibody is frequently used in combination with CD1a; the profile of Langerhans cell histiocytosis is CD207 and CD1a positive. CD207 expression is cytoplasmic. It is also critical to the identification of indeterminate cell tumors (e.g., histiocytic/dendritic), which shows expression of CD1a and S100, but lack CD207 expression.
BRAF V600E
BRAF (NS7, RAFB1) is a proto-oncogene that regulates cell division, differentiation, and secretion. It is located at 7q34. BRAF V600E is a mutation found in increasing numbers of malignancies (e.g., Langerhans cell histiocytosis, multiple myeloma, melanoma, colorectal carcinoma, papillary thyroid carcinoma, non-small cell lung carcinoma, and gliomas). Mutational status can predict prognosis as well as guide treatment in the form of small molecule inhibitors of BRAF. An immunohistochemical stain is used as a surrogate marker for the DNA based assays for this mutation. High sensitivity (98%) and specificity (97%) have been reported on diverse tumor types using automated platforms with monoclonal VE1. The staining pattern is granular-cytoplasmic.
Tryptase
Tryptase (tryptase α/β1) belongs to a family of serine proteases in which the alleles show so much sequence variation that they were once thought to be two separate genes: α and β1. β Tryptases appear to be the main isoenzymes expressed in mast cells; whereas in basophils, α-tryptases predominate.
Normal Bone Marrow: Immunohistochemical Identification of Normal Components
Normal bone marrow consists of a heterogeneous population of cells proceeding along various differentiation pathways. Although most cell types can be easily distinguished on bone marrow aspirate smears and biopsy sections of appropriate thickness, immunohistochemistry (IHC) is valuable in identifying specific cell subsets and in assessing their proliferative capability.
Erythropoiesis
The erythroid precursors typically account for approximately 30% of the nucleated cells in normal bone marrow. The erythroid cells can be identified by immunohistochemical staining of biopsy sections, with a number of antibodies targeting more mature and anucleated forms (glycophorin A, hemoglobin) and earlier precursors (CD71, E-cadherin). The earliest identifiable erythroid cell is the proerythroblast. In the erythroid series, cell division occurs down to the stage of polychromatophilic erythroblast. Normally, 
of the erythroblasts in the adult bone marrow express the proliferation associated marker Ki67.
Granulopoiesis
Granulocytes account for approximately 60% of the nucleated cells in the normal bone marrow, and these can be identified by a variety of IHC stains. The most specific is myeloperoxidase. Myeloperoxidase is expressed in cells belonging to the neutrophilic and eosinophilic series and to a lesser extent by monocytes ( Fig. 5.1 ). Staining for CD33 has comparable sensitivity and specificity in current paraffin antibodies. CD34, CD117, and HLA-DR are expressed by myeloblasts, with downregulation of those markers as they mature to the promyelocyte stage. Cells belonging to the later stages of neutrophilic differentiation can also be recognized by CD15 ( Fig. 5.2 ). CD10 will also stain a subset of mature neutrophils. CD45RO, CD74, and BCL2 also stain myeloid cells in marrow sections but are nonspecific. The epitopes of CD68 (PG-M1, HAM-56, and KP-1) react with myeloid cells as well as macrophages. PG-M1 is the most specific for macrophages/monocytes. The other two antibodies may be used as secondary markers for myeloid cells (see section on macrophages). Lysozyme is also a useful marker for myeloid cells, but less specific than myeloperoxidase and often has high background staining. Proliferation associated antigens, such as Ki67, are positive in myeloid cells to the metamyelocyte stage. CD43 stains almost all myeloid elements including AML but also stains macrophages and mature T cells and B and T-ALL. Basophils and eosinophils are usually identified by their morphologic and histochemical characteristics. However, immunohistochemical stains for basophils (2D7, BB1) and eosinophils (eosinophil major basic protein) exist but are only rarely applicable to diagnostic efforts.
CD163 and CD14 are highly specific and sensitive for mature macrophages but are only rarely seen in immature forms in acute leukemia. CD4 expression is seen in most monocytes/macrophages and in most AML with monocytic differentiation. MAC 387 and HAM56 stain myeloid cells, including a significant percentage of AML, but are not considered highly specific. The normal mast cell shows reactivity with tryptase, CD117, and CD68.
Megakaryopoiesis
Megakaryocytic progenitor cells express CD34 and HLA-DR. The maturation process of the megakaryocytic series is characterized by repeated endomitosis, with the generation of large cells with lobulated nuclei demonstrating 8, 16, or 32 ploidy. Megakaryocytes can be identified in tissue sections by their positivity with CD42b, factor VIII antigen, CD61, CD4, and CD31 ( Fig. 5.3 ). CD79a may be positive in megakaryocytes depending on the antibody used. Although these markers are more strongly expressed in mature megakaryocytes, they can also be used to identify immature cells of the megakaryocytic lineage. Staining of the megakaryocytes with Ki67 is variable and not particularly related to the nuclear segmentation of the cells. However, increased staining can be observed after treatment with thrombopoietic growth factors.
Lymphoid Cells
Lymphocytes account for up to about 20% of the nucleated cells in the normal bone marrow. However, higher values may be observed in older patients and in children. Usually T cells outnumber B cells by 4 : 1. Lymphoid follicles may also be observed, usually in adults, especially after the age of 50. The T cells can be identified by T-cell antibodies, which are reactive in routinely processed marrow (e.g., CD45RO, CD2, CD3, CD4, CD8, CD5, CD7, LEF1). In a normal marrow, the CD8 T-lymphocytes are more numerous than CD4 T lymphocytes. The marrow B cells are stained by PAX5, CD20, and CD79a. Although not entirely specific, patterns associated with malignant versus benign lymphoid aggregates include (1) predominance of B cells, (2) infiltrative borders, (3) paratrabecular location, (4) large size, and (5) cytologic atypia. Features more commonly associated with benign aggregates include predominance of T cells.
Rare PAX5, TdT, CD10, and CD79a positive early B-cell precursors (also termed hematogones) are normally present in the marrow. Hematogones are more numerous in infancy and early childhood, and numerical comparisons will not allow distinction from ALL. In ALL, however, the blasts usually consistently express CD34 and TdT, whereas in normal marrows, the number of TdT positive cells is higher than the number of CD34 positive cells. In nonneoplastic marrows, CD34 positive cells are present singly and usually account for less than 2% of the marrow nucleated cells.
Scattered NK cells can be recognized in marrow sections by CD56 and sometimes CD57. CD56 also highlights osteoblasts lining the bone trabeculae.
Bone Marrow Stroma
The extracellular matrix, which can be demonstrated in routine bone marrow biopsy sections, includes reticulin (immature collagen) and collagen IV. Bone marrow reticulum cells can be identified by nerve growth factor receptor (NGFR) positivity. Vascular endothelial cells can be stained by ERG, CD34, WT1, and CD31. Differences in staining pattern can be observed with these antibodies (e.g., some sinusoids are negative with CD34).
Myeloid Diseases
The most commonly encountered myeloid diseases can be separated into three general categories: MDSs, myeloproliferative neoplasms (MPN), and AML. Although there is significant heterogeneity, entities within these groups share a number of important clinical, biologic, and diagnostic features, discussed later. The current WHO classification identified many subgroups based on clinical, genetic, immunophenotypic, and morphologic features. Complete discussion of the classification is beyond the scope of this discussion and is better addressed in specialized texts. Although the role of cytogenetic and molecular testing is critical in the evaluation of these malignancies, morphologic and immunohistochemical assessment remains important for classification and risk stratification, especially in cases where samples are not available for ancillary testing such as flow cytometry or genetics.
Myelodysplastic Syndromes
MDSs are a heterogeneous group of clonal myeloid neoplasms characterized by ineffective hematopoiesis and peripheral blood cytopenias. Although the natural history is variable, all the MDSs are associated with a defined risk of progression to AML. As such, these neoplasms were historically referred to as preleukemia. Accurate classification allows for effective risk stratification, prognostication, and treatment. Although classically considered a disease of older adults, childhood MDS is also identified.
MDS arises from a clonal expansion of an abnormal hematopoietic stem cell that retains some of the capacity to morphologically mature in the marrow space but shows ineffective terminal maturation. Irrespective of number or degree of cytopenia or preservation of maturation assessed morphologically, by the time the disease manifests clinically, the vast majority of marrow cellularity represents progeny of the malignant stem cell. Undoubtedly, a growth and survival advantage is gained by the proliferating malignant cell, but there is a paradoxical increase in apoptosis. Approximately half of cases present with recurrent cytogenetic abnormalities by routine analysis. Although these chromosome-level changes are thought to be a secondary event, they play an increasingly important role in risk stratification and provide a means of guiding therapy. When evaluated by methods such as massively parallel sequencing (next-generation sequencing [NGS]), many cases without overt cytogenetic abnormalities are shown to have mutations in genes associated with myeloid maturation, proliferation, signaling, tumor suppressors, and epigenetic regulators. The prognostic impact of cytogenetic abnormalities is clinically accepted and reflected in the Revised International Prognostic Scoring System for MDS. The degree of cytopenia, blast count, and karyotypic abnormalities are assigned scores that are significantly associated with median survival and evolution to AML.
MDS is usually associated with topographic alterations in the bone marrow, and bone marrow biopsy can be used to provide useful diagnostic and prognostic information in these disorders. A prognostically important morphologic finding in MDS is the presence of aggregates of immature myeloid cells in an abnormal central marrow cavity location. This abnormal localization of immature precursors (ALIP) or cluster of CD34 positive blasts are mainly present in the aggressive MDS subtypes and are associated with a poor prognosis and an increased incidence of progression to acute leukemia. Presence of ALIP, however, is not unique to MDS and has been reported in reactive hematologic conditions (e.g., status post bone marrow transplantation and post induction chemotherapy). In addition, the identification of the presence of ALIP may be compromised by using paraffin sections of excessive thickness or otherwise suboptimal morphology. CD34 can be used as a surrogate marker for the presence of ALIP. Both an increase in the percentage of CD34 positive cells and a tendency of positive cells to form aggregates have been shown to be reliable predictors of leukemic transformation and of survival in MDS cases. The occurrence of large sheets of CD34 positive blasts can be proposed as a means of recognizing MDS undergoing transition to AML. Immunostaining for CD34 is especially important in two subsets of patients with MDS: MDS with fibrosis (MDS-f) and MDS with hypocellular marrow (MDS-h). Immunostaining for CD34 in megakaryocytes can be seen in myeloid disease but is not sensitive or specific ( Fig. 5.4 ).
It should be noted that aberrant expression of antigens of maturing myeloid elements, such as CD7, can be identified by flow cytometry and may be present by immunohistochemistry. However, aberrant antigen expression is not specific for a diagnosis of MDS, and although this finding by immunohistochemistry may be supportive of a neoplastic myeloid disorder, it would not be useful in subclassification.
Myelodysplastic Syndrome With Fibrosis
MDS-f is commonly high grade and as aspirates are typically hemodilute, and an assessment of blast count by CD34 or CD117 is imperative. The use of antibodies reactive with megakaryocytes has shown that these patients have a higher number of these cells than either healthy subjects or patients affected by MDS without fibrosis. Furthermore, primary and secondary MDS-f, although clinically and histopathologically similar, differ in terms of the number of megakaryoblasts, which are significantly higher in the primary forms. MDS-f needs also to be distinguished from primary myelofibrosis (PMF; dysplasia only in megakaryocytes, giant megakaryocytes, prominent splenomegaly, tear drops erythrocytes) and therapy-related MDS, an aggressive MDS, which is often characterized by variably cellular and fibrotic marrows.
Hypocellular Myelodysplastic Syndrome
CD34 can be used to distinguish hypoplastic MDS from acquired aplastic anemia. The former disorder is characterized by high CD34 expression as compared with aplastic anemia.
Myeloproliferative Neoplasms
MPN represent a group of myeloid diseases characterized, at least at some point of their natural history, by increased peripheral blood counts and bone marrow hypercellularity. As is the case with most myeloid tumors, we have gained a tremendous understanding of the underlying genetic changes that affect classification and prognostication of these neoplasms. The quintessential MPN, chronic myeloid leukemia (CML), BCR-ABL1-positive is defined by its single genomic aberration, which is necessary for diagnosis, targeted therapy, and disease monitoring. More recently, molecular changes have been identified that help with classification of the other MPN, namely, polycythemia vera (PV), essential thrombocythemia (ET), and PMF. However, because none of these changes is specific, diagnosis remains dependent on morphologic evaluation of the bone marrow, often aided by immunohistochemistry.
Chronic Myeloid Leukemia, BCR-ABL1-Positive
CML is a biphasic or triphasic disease with invariable progression to a blast phase without therapeutic intervention. Because the defining translocation t(9;22) is present in an early undifferentiated precursor, additional molecular changes can manifest as either AML or ALL.
Although many patients present in the chronic phase of disease, assessing the degree of bone marrow fibrosis and assessing blast count are important prognostic and predictive markers. Increased bone marrow fibrosis is associated with increased blast count and karyotypic abnormalities and may affect hematopoietic reconstitution after bone marrow transplantation. Blast counts are important features of progression from chronic to accelerated phase, and ultimately blast phase of disease. Although these are best assessed by quality bone marrow aspirate preparations, marrow fibrosis or hemodilute smears may require ancillary immunohistochemical assessment, including CD34 and TdT. Myeloid blasts greater than 20% of marrow cellularity define the AML blast phase of disease, whereas greater than 5% lymphoid blasts define the lymphoid blast crisis.
Essential Thrombocythemia, Polycythemia Vera, Primary Myelofibrosis
Immunohistochemical evaluation of MPN can be of benefit in select cases of non-CML MPN. In these cases, CD34 and CD117 may be useful in enumerating blasts. In PMF, when significant fibrosis is present, aspirates may not be available to quantify blasts. Increases in blasts can support both accelerated phases of non-CML MPN, and if sheets of blasts of more than 20% are identified, then this would support a diagnosis of a blast phase. The use of megakaryocytic markers may be of benefit in some cases; some MPN may have such unusual megakaryocytes as to require confirmation of their identity, in contrast to a nonhematopoietic neoplasm or highly pleomorphic lymphoma. Recently, an immunohistochemical stain for mutant calreticulin (CALR) antibody has been developed. If present, the expression would confirm the presence of mutant CALR, confirming a diagnosis of a myeloproliferative neoplasm, PMF, ET or MPN, unclassifiable, because CALR mutations are not seen in CML or PV.
Myelodysplastic Syndrome/Myeloproliferative Neoplasms
MDS/MPN are a heterogeneous group of neoplasms with a common feature: defective but excessive proliferation. Although a number of neoplasms fall within this category, MDS/MPN notably includes CMML, juvenile myelomonocytic leukemia (JMML), and atypical chronic myeloid leukemia (aCML). Bone marrow blast count, highlighted by CD34, is an important prognostic indicator because blast counts greater than 20% are diagnostic of AML. In cases of JMML and CMML, the marrow may also contain an increased number of monocytic cells reactive with CD68, and sometimes CD163 and CD14. The differential diagnosis of JMML is based on a multiparametric approach that includes the assessment of morphologic and genetic findings. In the work up of JMML, bone marrow biopsy can be very useful by excluding an infection associated hemophagocytic syndrome in patients with low white blood cell (WBC) counts and organomegaly or in ruling out acute leukemia. The blast cells in JMML express CD34 and are only rarely positive with myeloperoxidase. A proportion of the cells express CD68 (PG-M1) and lysozyme.
Acute Leukemias
The morphologic analysis of acute leukemia begins with the examination of Wright-Giemsa–stained smears of peripheral blood and bone marrow aspirates. Although the cytogenetic and molecular features are vital in the classification of AML, the immunophenotypic features of disease, assessed by flow cytometry or immunohistochemistry, are necessary for diagnosis ( Box 5.1 ). Morphologic and immunohistochemical assessment are particularly important when interpreting bone marrow biopsy material in a patient with “dry tap” marrow aspirates as well as extramedullary leukemic infiltrates.
CD15 is not sensitive by IHC in AML.
CD14 is not sensitive for monocytic differentiation in AML by IHC.
CD163 is not sensitive for monocytic differentiation in AML by IHC.
CD45 may be expressed weakly or absent by IHC in acute leukemias including AML.
Expression of myeloperoxidase and/or CD33 by IHC is specific but not sensitive for AML if staining is identified in blasts.
Aberrant T/NK cell antigen expression (CD56>CD7>CD2>CD5) can be seen in AML.
CD10 expression is not seen in AML, although it can be identified in mature granulocytes.
Expression of CD34 is only seen in about 50% of AML by IHC staining.
CD4 expression in AML supports monocytic differentiation.
PAX5 expression in AML is commonly seen in AML with t(8;21) but is also seen in B-ALL.
Tdt and CD99 expression can be seen in a subset of AML; it is not helpful in distinguishing AML from ALL.
The lack of CD34 and HLA-DR can be demonstrated in acute promyelocytic leukemia. However, the lack of expression of these markers in AML by IHC is not specific and can be seen in non-APL subtypes of AML.
AML, Acute myeloid leukemia; IHC, immunohistochemistry.
Acute Myeloid Leukemias
AML morphologically manifests as a proliferation of immature myeloid precursors, typically accounting for more than 20% of marrow cellularity ( Fig. 5.5 ). A number of immunohistochemical stains are helpful to provide diagnostic information and subclassification.
Identification of cell lineage is fundamental in the diagnosis of AML. However, when the sample is identified as AML, current classification is heavily dependent on the presence or absence of cytogenetic or molecular abnormalities for subclassification. Although immunophenotypic evaluation may provide some insights into classification, especially in cases without known genetic or molecular abnormalities, classification with cytogenetics, fluorescence in situ hybridization (FISH), or more advanced techniques should be attempted if appropriate samples are available.
As mentioned previously, identification of blasts can be accomplished with markers such as CD34, CD117, CD99, or TdT. CD34 is only positive in 50% of AML, so it is not optimal, but when positive, it can be used to enumerate blasts in a paraffin-embedded sample. CD117 can be seen on some other cell types, including normal immature early erythroid precursors and mast cells. CD33, CD68, and myeloperoxidase antibodies are both sensitive and specific for identification of myeloid lineage and, if expressed in cells that are known to be blasts, would support a diagnosis of AML. Other, less specific markers include lysozyme. HLA-DR is expressed on most cases of AML but is notably absent in acute promyelocytic leukemia. Acute promyelocytic leukemia also typically lacks expression of CD34 and may express CD56.
Monocytic differentiation may be difficult to characterize by immunohistochemistry ( Fig. 5.6 ). Monocytes will express CD68 but are typically negative for myeloperoxidase. CD4 is positive in many cases. They will also express lysozyme and HLA-DR, which lack specificity. Although CD14 and CD163 are monocytic/histiocytic lineage specific markers, they are uncommonly positive in the immature blasts of monocytic leukemias.
