Immunohistology of Lymph Node and Lymph Node Neoplasms

CD20

CD20 is a 35-kDa nonglycosylated membranous phosphoprotein. CD20 expression is acquired by the late pre-B-cell stage of development and is typically lost at the plasma cell stage of terminal differentiation. Although the exact function of the CD20 antigen is unknown, it is thought to be involved in B-cell regulation, differentiation, and calcium flux. L26 is the most commonly used commercial antibody. CD20 staining is membranous.

Currently, CD20 is the first-line B-cell lineage defining antibody. Therapeutic strategies involving the monoclonal antibodies directed against CD20 (i.e., rituximab containing chemotherapeutic regimens) have necessitated the use of alternative B-cell lineage defining markers in patients who have received such therapy and are suspected of relapse. Although rarely identified, CD20 expression has been noted on a small subset of nonneoplastic T cells. CD20 expression has is identified in occasional cases of Hodgkin lymphoma, precursor B acute lymphoblastic leukemia, plasma cell neoplasms, and rarely in T-cell lymphomas.

PAX5 (see later), CD79a (see later), CD19, and CD22 are pan-B-cell antigens also available for staining in paraffin sections and are useful adjuncts to determine B-cell lineage in patients who have received treatment with rituximab or other anti-CD20 agents.

PAX5

PAX5 (B-cell–specific activator protein) is a nuclear protein belonging to the paired-box containing (PAX) family of transcription factors. PAX5 is thought to commit B-cell progenitors to the B-cell lineage by suppressing non–B-cell associated genes and activating a B-cell–specific program. ^, A broader regulatory role has been described, including regulation of cell adhesion and migration, inducing V-DJ immunoglobulin heavy chain recombination and facilitating early B-cell receptor signaling to promote development to the mature B-cell stage. The staining pattern in B cells is strong and nuclear; Reed-Sternberg cells show a characteristic weak/variable pattern of nuclear expression.

PAX5 is expressed by early B-cell precursors as well as mature B cells, and it is lost as B cells mature to plasma cells. Expression of PAX5 on normal B cells as well as their malignant counterparts has become a valuable marker of B-cell lineage. Rare reports of PAX5 expressing non–B-cell lineage neoplasms have been described in anaplastic large cell lymphoma (ALCL) and lymphomatoid papulosis. Expression occurs in non-hematolymphoid malignancies, as PAX5 expression has been identified in atypical carcinoids, small cell lung carcinomas, and large cell neuroendocrine carcinomas.

CD79a

CD79a is associated with the immunoglobulin receptor complex in the B-cell membrane. It is the B-cell marker with the broadest sensitivity as it is expressed in early B-cell precursors (even before immunoglobulin heavy chain gene rearrangement), throughout B-cell maturity, and is eventually lost only in the latest plasma cell stage. The staining pattern is cytoplasmic.

Although a B-cell lineage antigen, CD79a expression has also been reported in cases of T-cell lymphoblastic lymphoma and acute myeloid leukemia (often in acute promyelocytic leukemia). ^,

BCL6

The BCL6 gene encodes a 79-kDa zinc finger binding protein thought to play a role in B-cell differentiation within the germinal center. BCL6 is expressed on B cells of germinal center origin with a nuclear staining pattern.

Expression of BCL6 protein, demonstrated through IHC, does not necessarily correlate with the presence of a bcl6 gene rearrangement. Rearrangements of bcl6 are one of the more common genetic abnormalities and are found in diverse lymphomas, not restricted to those arising from a B-cell germinal center origin. In contrast, BCL6 protein is normally expressed nonneoplastic germinal center B cells (GCBs). Lymphomas of germinal center origin, such as follicular lymphoma (FL) or Burkitt lymphoma (BL), express BCL6 protein demonstrated by IHC; however, this does not imply the presence of a BCL6 translocation. Although more often seen in cells of the B-cell lineage, BCL6 expression is reported in occasional ALCLs and peripheral T-cell lymphomas (PTCLs). ^,

Other makers that identify germinal center origin are GCET1, HGAL/GCET2, and LMO2. These markers are used in various classifying systems for determining GCB versus nongerminal center B (NGC) origin of diffuse large B-cell lymphomas (DLBCLs).

Multiple Myeloma Oncogene-1/Interferon Regulatory Family 4

Multiple myeloma oncogene-1 (MUM1) belongs to a larger group termed the interferon regulatory family (IRF4) and encodes a transcription factor responsible for development in B, T, plasma, dendritic, and myeloid cells. There are two commercially available antibodies against human MUM1/IRF4 protein, Mum-1p (monoclonal) and ICSTAT (polyclonal). Staining is both nuclear and cytoplasmic.

MUM1 was originally recognized by its upregulation in multiple myeloma with t(6;14)(p25;q32); however, it was soon found not to be specific for plasmacytic differentiation. MUM1 protein expression has been described in numerous neoplasms including lymphoplasmacytic lymphoma (LPL), chronic lymphocytic leukemia (CLL), FL, marginal zone lymphoma (MZL), DLBCL, primary mediastinal large B-cell lymphoma (PMLBCL), primary effusion lymphoma (PEL), BL, Hodgkin lymphoma, ALCL, PTCL-not otherwise specified (NOS), adult T-cell lymphoma/leukemia, and melanoma. ^, MUM1 expression is also seen in nonneoplastic “activated” T cells, a subset of GCBs, and normal melanocytes. ^, In general, MUM1 immunostaining is thought to parallel that of CD30 staining. ^, Translocation of IRF4 is seen in a subset of cutaneous ALCL and a small subset of other T-cell lymphomas. The expression of MUM1/IRF4 can be seen in many T-cell lymphomas and be independent of the presence of the translocation. A rare pediatric lymphoma with features of DLBCL or FL, occurring mostly in the Waldeyer ring, has been identified. In contrast to most FLs, these express MUM1, and a substantial portion of these cases have IRF4/MUM1 translocations.

OCT2 and BOB1

OCT2 and its coactivator BOB1 are transcription factors of the POU homeodomain family that bind to the conserved octamer sites in the promoters of the immunoglobulin genes involved in B-cell differentiation and regulation. ^, Staining is nuclear.

BOB1 is a novel 256-amino acid proline-rich protein that is seen predominantly in the B-cell lineage expressed in precursor and mature B cells. BOB1 interacts with either OCT1 or OCT2 to coactivate gene transcription by binding to a number of octamer sites throughout the genome. BOB1 is also called OBF1, OCT Binding Factor 146, OCA-B47, or Bob-148.

Given the postulated mechanisms of action and origins of classic Hodgkin lymphoma (CHL), OCT2 and BOB1 expression were thought to be absent in Reed-Sternberg cells, a fact which was largely used to differentiate the LP (“popcorn cells”) of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) from Reed-Sternberg cells of CHL. However, weak expression of OCT2 or BOB1 in the Reed-Sternberg cells of CHL has been reported in a subset of cases. ^, Strong consistent expression of OCT2 and BOB1 is reported in NLPHL and in NHLs. OCT2 and BOB1 are also expressed in a subset of acute myeloid leukemias, where their expression may have a prognostic relevance.

CD138

CD138 (syndecan-1) is a 200-kDa member of the transmembrane family of heparin sulfate proteoglycan proteins. Located on chromosome 2p23-24, CD138 is thought to be a receptor for matrix proteins and a cofactor for growth factors. ^, The anti-syndecan 1 monoclonal antibody Mi15 is frequently used. The staining pattern is membranous.

CD138 is most commonly associated with plasmacytic differentiation and is seen on both benign plasma cells and their malignant counterparts (plasma cell myeloma [PCM]). Among hematolymphoid neoplasms, expression of CD138 is interpreted as evidence of plasmacytic differentiation; however, caution should be used as NHLs and many non-hematolymphoid neoplasms may express this marker. CD138 expression is seen on nonneoplastic epithelial surfaces and correspondingly has been reported among various types of carcinomas. Nonneoplastic early precursor B cells and posttransplant lymphoproliferative disorders express CD138. Plasmablastic lymphoma, LPL, and a small subset of cases of CLL express CD138. Expression of CD138 on mesenchymal neoplasms and tumors of melanocytic origin have also been reported.

CD30

CD30 is a membrane bound phosphorylated glycoprotein weighing 120 kDa. It is a member of the tumor necrosis factor (TNF) receptor superfamily 8 (TNFRSF8). ^, Monoclonal antibodies used in paraffin include Ber-H2, HeFi, Hodgkin/Reed-Sternberg (HRS)-1, HRS-2, HRS-3, M44, M67, and C10. CD30 is expressed on normal activated T and B cells and virally transformed B and T cells (Epstein-Barr virus [EBV], human T-cell lymphotropic virus-1 or -2, and human immunodeficiency virus [HIV]). Monocytes/macrophages and granulocytes also constitutively express CD30. In lymph node and tonsil sections, a small subset of lymphocytes in the parafollicular areas express CD30. CD30 is thought to transduce a cell survival signal and be involved in the T-cell–dependent portion of the immune response. ^, CD30 staining may be membranous or concentrated in the Golgi zone outside the nucleus (paranuclear).

CD30 is consistently over-expressed on HRS cells. However, CD30 expression is seen in several settings including lymphomatoid papulosis, ALCL, adult T-cell lymphomas, some cutaneous T-cell lymphomas, natural killer (NK) neoplasms, a subset of B-cell lymphomas (DLBCL, BL), and embryonal carcinoma. CD30 expression in neoplasms has become of increasing importance with the development and successful treatment with anti-CD30 monoclonal antibodies. Brentuximab vedotin is an anti-CD30 chimeric IgG1 monoclonal antibody used to target CD30 neoplasms particularly in the setting of a relapse after first line therapy has failed.

ALK

Anaplastic lymphoma kinase (ALK) is a 220-kDa tyrosine kinase receptor belonging to the insulin receptor superfamily. Initially described in ALCL, the t(2;5)(p23;q35) created a fusion gene from the nucleophosmin gene and the tyrosine kinase receptor gene (ALK) , and the resulting chimeric gene encoded a constitutively activated tyrosine kinase that is a potent oncogene. Additional translocations were subsequently discovered; at least 15 different ALK fusion proteins have been described. Although initially described in ALCL, numerous other malignancies express ALK either as activated fusion proteins derived from chromosomal rearrangements or as mutationally activated ALK proteins (such as the activating mutations described in neuroblastoma). Staining may be cytoplasmic, nuclear, or membranous, and different staining patterns correlate with specific translocations.

Normal ALK immunohistochemical expression is seen in rare scattered neural cells, endothelial cells, and pericytes in brain of adults. ALK expression has been reported in cases of B-cell lymphomas, non–small cell lung cancer (NSCLC), rhabdomyosarcomas, glioblastomas, melanomas, inflammatory myofibroblastic tumors, esophageal squamous cell carcinomas, and systemic histiocytosis. Crizotinib (PF-02341066), a receptor kinase inhibitor, has been used to treat ALCL and NSCLC cell lines that harbor ALK translocations. Ceritinib and alectinib are newer ALK inhibitors with several others currently in development. The ALK1 antibody is used in lymphoma diagnosis, whereas other highly sensitive antibodies (including D5F3, 5A4) are used to detect positive lung cancer and other nonhematopoietic tumor cases. Although the highly sensitive antibodies can be used for lymphoma and hematologic cases, the ALK1 antibody is not sensitive enough to use in the nonhematologic tumors.

Terminal Deoxynucleotidyl Transferase

Terminal deoxynucleotidyl transferase (TDT) is a DNA polymerase that catalyzes the addition of deoxynucleotides to the 3′-hydroxyl terminus. ^, TDT is expressed in precursor B and precursor T lymphocytes during early differentiation; it generates antigen receptor diversity in both cell lines by synthesizing non-germline elements at the ends of rearranged immunoglobulin heavy chain and T-cell receptor (TCR) gene segments, respectively. Staining may be nuclear and membranous or with paranuclear dot-like positivity.

Physiologic TDT expression is seen in sections of the thymus containing precursor T cells, and in bone marrow sections containing early B-cell precursors, so-called hematogones. The malignant blasts of acute T or B lymphoblastic leukemia/lymphoma express TDT and a subset of acute myeloid leukemias and hematodermic CD56+/CD4+ neoplasms. Nonhematopoietic malignancies expressing TDT include some pediatric small round blue cell tumors, Merkel cell carcinoma, and small cell lung carcinoma.

CD43

CD43 is a sialomucin expressed on hematopoietic precursors and is thought to play a role in regulation of hematopoiesis. In adults, CD43 occurs both on bone marrow hematopoietic stem cells and on mature white blood cells in the periphery with the exception of resting B lymphocytes. CD43 is also found on tissue macrophages, dendritic cells, smooth muscle cells, epithelium, and endothelium. CD43 expression has been reported on myeloblasts, lymphoma cells, and metastases of solid neoplasms. The aberrant expression of CD43 on B cells can be useful in identifying B-cell lymphomas. Staining is cytoplasmic.

Cyclin D1

Cyclin D1 (BCL1/PRAD1/CCND1) is a transcriptional regulator protein that complexes with the cyclin dependent kinases that maintain regulation of G1 to the S phase of the cell cycle. ^, The CCND1 gene is found at chromosome 11q13. Most notably, mantle cell lymphoma (MCL) is associated with the translocation involving this gene and the immunoglobulin heavy chain locus: t(11;14)(q13;q32). In addition to MCL, a subset of cases of PCM and hairy cell leukemia (HCL) have been found to express cyclin D1. Focal areas of weak cyclin D1 expression have been described in the proliferation centers of CLL. Some cases of DLBCL express cyclin D1 without any significant impact on prognosis. Nonneoplastic lymphoid cells do not express cyclin D1. Over-expression of cyclin D1 is not restricted to hematopoietic malignancies and has been associated with breast carcinoma and NSCLC. Scattered nonneoplastic endothelial cells will often express cyclin D1 and can be used as an internal positive control. Staining is nuclear.

BCL2

Immunohistochemical antibodies detect an increase in the antiapoptotic BCL2 protein, often resulting from the translocation of the BCL2 gene to a position behind the enhancer elements of the Ig heavy chain gene t(14;18)(q32;q21). In addition to inhibiting apoptosis, over-expressed BCL2 may also block chemotherapy-induced cell death. Staining is membranous.

BCL2 expression may be used as one means of differentiating benign follicular hyperplasia from the neoplastic counterpart FL; however, BCL2 expression should not be interpreted as evidence of malignancy. Intrafollicular T cells, T and B cells in the interfollicular areas, primary follicles, and mantle zone B cells normally express BCL2. Expression of BCL2 is not limited to lymphomas and is commonly encountered in nonhematopoietic malignancies as well. Loss of BCL2 expression may be seen in T-cell lymphomas.

CD2

CD2 is a 50- to 55-kDa transmembrane glycoprotein that is found on both T cells and NK cells. ^, The CD2 genes are found on chromosome 1 and are thought to play a role in antigen independent adhesion and in T-cell activation. ^{, ,} In T-cell development, it is thought to appear after CD7. The staining is cytoplasmic.

CD2 expression is seen on both immature (precursor) T cells and mature (peripheral) T cells. Aberrant loss of CD2 expression is seen in a subset of T-cell lymphomas. CD2 expression is usually seen in T acute lymphoblastic leukemia and more rarely may be seen on the myeloblasts of acute myeloid leukemia. CD2 expression in mast cells is considered aberrant and supportive of a diagnosis of mastocytosis.

CD3

CD3 is a T-cell antigen composed of four distinct subunits (ε, γ, δ, and ζ) that span the membrane and are associated with the TCR and is considered a first-line antigen in identification of T-cell lineage. ^, CD3 staining is membranous and cytoplasmic.

CD3 is first found in the cytoplasm of developing T cells, cytoplasmic CD3 (cCD3). As T cells mature, the CD3 antigen moves to the surface. Similarly, the neoplastic counterparts show a similar distinction with cytoplasmic CD3 on precursor T-cell neoplasms and surface CD3 seen on peripheral “mature” T-cell neoplasms. Although most widely used as a T-cell lineage specific antigen, CD3 expression (cytoplasmic and membranous) has been reported on B-cell lymphomas, particularly those with expression of EBV.

CD4/CD8

CD4 and CD8 T-cell surface molecules play a role in T-cell recognition and activation by binding to their respective class II and class I major histocompatibility complex (MHC) ligands on an antigen-presenting cell (APC). CD4 has the additional role of stabilization of the TCR complex; it is also a major target of HIV. Staining for both CD4 and CD8 is both cytoplasmic and membranous.

Early T-cell precursors express both CD4 and CD8 simultaneously, and then with maturation lose one of these markers. CD4 staining is seen in the T-helper cells, the predominant population in the T-cell compartment. CD8 staining is seen in the cytotoxic T-cell population and nonneoplastic sinusoids of the spleen. The majority of T-cell lymphomas express CD4, not CD8, and are thought to be derived from the T helper lineage. Cytotoxic T-cell lymphomas expressing CD8, not CD4, are a proportionally smaller group of lymphomas. Rarely, lymphomas may have aberrant loss of both these antigens; however, a small subset of nonneoplastic T cells will also be “double negative” for these two markers, the γ/δ T cells, and this should not be misinterpreted as aberrant antigen loss.

CD5

CD5 is a 67-kDa type I glycoprotein that is thought to attenuate signals arising from the cross-linking of the TCR and its antigen on the MHC of APCs. ^, Staining is membranous.

CD5 is expressed by the majority of peripheral (or mature) T cells; the loss of this marker may be seen as one of the first findings in a developing T-cell lymphoma. ^, However, this finding has also been reported in reactive populations of T cells. Expression of CD5 has also been used to distinguish the neoplastic thymocytes of thymic carcinoma from a benign thymoma. Although a T-cell lineage antigen, CD5 is famously co-expressed aberrantly on certain B-cell lymphomas: CLL/small lymphocytic lymphoma (SLL) and MCL. More rarely, cases of MZL, DLBCL, and cases of FL may express this T-cell antigen as well. In addition, a small nonneoplastic subset of B cells, the B1a cells, normally expresses this T-cell marker.

CD7

CD7 is a 40-kDa glycoprotein member of the immunoglobulin gene family. CD7 antigen is thought to be involved in signal transduction, proliferation, and adhesion. The CD7 monoclonal antibody, CBC.37, shows expression on the majority of peripheral T cells (i.e., mature T cells), NK cells, and precursor T cells (immature T cells). Staining is membranous.

CD7 is one of the earliest markers in T-cell development. Although a T-cell lineage antigen, CD7 expression may be seen in small populations of fetal bone marrow B cells, and myeloid precursor cells; however, this is subsequently lost early with differentiation. As with CD5, loss of CD7 antigen may be seen in the setting of a T-cell lymphoproliferative process as well as in a benign reactive setting. Aberrant CD7 expression has been described in the myeloblasts of acute myeloid leukemia, where it has been associated with Fms-like tyrosine kinase-3 internal tandem duplication (FLT3/ITD) mutation, and significantly shorter disease-free/postremission survival. ^,

T-Cell Intracytoplasmic Antigen1/Granzyme B/Perforin

The T-cell compartment is composed of CD4 expressing T helper cells, and CD8 expressing T cytotoxic cells. T-cell intracytoplasmic antigen (TIA1), granzyme B, and perforin are markers used to identify the cytotoxic T cells that induce lysis of their targets by using these granule-associated cytotoxic proteins. These cytotoxic markers can also be seen in NK cells and granulocytes. Expression of TIA1 can be detected in all cytotoxic cells, whereas granzyme B and perforin expression can be detected in high levels only in activated cytotoxic cells. Expression is cytoplasmic.

CD56/CD57

CD56 and CD57 are frequently used to identify the NK cell lineage; however, they are frequently found expressed in other, nonhematopoietic tissues. CD56, or neuronal cell adhesion molecule (NCAM), is a NK cell marker that is also expressed in the central and peripheral nervous systems. Most cases of PCM (∼70%) also express this marker. Similarly, CD57 is a marker expressed on NK cells as well as other T cells; it is occasionally used in the diagnosis of NLPHL to visualize the small T cells ringing the LP cells. The staining of CD56 (monoclonal antibody 123C3) and CD57 are membranous.

NK cells express CD2, CD7, CD8, CD56, and CD57. They are positive for cytoplasmic CD3, but they not surface CD3 and do not typically express CD5. The neoplastic counterpart: extranodal NK/T-cell lymphomas express CD2, cytoplasmic CD3, CD56, and in most cases EBV. ^,

Additional markers for NK cells include killer inhibitory receptors (KIRs). KIR expression is identified using monoclonal antibodies specific for CD94, CD158a, and CD158b.

Beta F1

βF1 staining identifies a portion of the β subunit of the T cells that carry the α/β TCR. T cells with the α/β TCR normally represent the majority of T cells (i.e., 95%). Similarly, the majority of T-cell lymphomas express the α/β TCR. Staining is membranous.

Negativity for βF1 stain implies that the T cells carry the alternative TCR subunits: γ/δ. Small subsets of normal γ/δT cells are found in the skin, splenic red pulp, mucosal associated lymphoid tissue (MALT), and in the medulla of the thymus. γ/δ T cells have a distinct immunophenotypic profile so that CD2 and CD3 are positive but CD4 and CD8 are negative (double negative T cells), and CD5 is likewise usually negative. The neoplastic counterparts also express the γ/δ TCR: hepatosplenic T-cell lymphoma and subcutaneous panniculitic type T-cell lymphoma (SPTCL). However, caution should be used in interpreting this negative staining as evidence of a γ/δ T-cell derivation, as NK cells also are negative for β F1, staining with CD56 in this instance helps to identify these cells.

Immunohistochemical staining for the δ subunit has become available but is technically challenging. Its use is not widespread, but when appropriate, staining can confirm γ/δ origin in embedded tissues.

Lymphoid Enhancer-Binding Factor 1

Lymphoid enhancer-binding factor 1 (LEF1) is associated with a gene that encodes a transcription factor belonging to a family of proteins that share homology with the high mobility group protein-1. The protein can bind to an important site in the TCR-α enhancer, inducing enhancer activity. It is also involved in the Wnt signaling pathway and may function in hair cell differentiation and follicle morphogenesis. Mutations in this gene have been found in somatic sebaceous tumors. This gene has also been linked to other cancers, including androgen-independent prostate cancer.

LEF1 is a nuclear stain and staining can be variable in intensity, but any staining, even weak, is considered positive. Aside from hematopoietic cells, there are only a few positive cell types. Colon cancer, but not normal colon, shows expression, and pancreatic tumors and normal pancreas show expression. There is normal expression in hair follicles in skin/adnexal structures.

In hematopoietic tissues, LEF1 is a pan-T-cell antigen. It has the same distribution and number of cells positive as typical T-cell stains including CD3. LEF1 is aberrantly expressed in the majority of cases of CLL/SLL. It is positive in greater than 90% cases of CLL/SLL. It is not considered to be expressed in any significant number of non-CLL small B-cell lymphomas. A recent report suggests that LEF1 staining may be seen in 5% to 8% of cases of MCL. It is expressed on a subset of DLBCL and can be expressed in large cell (Richter) transformation of CLL.

Immunoglobulin D

Immunoglobulin (Ig) D is an immunoglobulin with a delta heavy chain present on the surface of B lymphocytes or as a soluble form in plasma. IgD and IgM are coexpressed on the surfaces of most peripheral B cells. IgD is normally expressed in resting mantle cells and can be useful in identifying primary follicles. In addition, the small dark blue lymphocytes in the nodules of NLPHL are typically of mantle cell type and are positive for IgD. IgD expression in LP cells has been reported in a subset (27%) of NLPHL cases with an extrafollicular distribution of LP cells, and a relatively T-cell–rich background. In contrast, IgD expression is rarely seen in T-cell/histiocyte–rich large B-cell lymphoma (TCHRLBCL).

PD1 (CD279) and PD-L1

Programmed cell death protein 1 (PD1; CD279) functions with its ligand programmed death-ligand 1 (PD-L1) as an immune checkpoint protein. PD-L1 is normally expressed on a subset of T cells and other cells of the immune system as well as seen on some malignant tumor cells (melanoma, lung, kidney, or bladder). As such, it has become a target for therapy even independent of immunohistochemical staining on tumor specimens. The pattern of immunoreactivity is membranous. A recent study demonstrated that the ligand of PD1, PDL1, is expressed in HRS cells of 70% evaluated CHL cases, 54% of NLPHL, and approximately a third of PMLBCLs and DLBCLs.

CD45

CD45 or leukocyte common antigen (LCA) is a 200-kDa transmembrane glycoprotein expressed on most hematopoietic cells, with the exception of erythroid cells and a subset of plasma cells. The protein is a member of the protein tyrosine phosphatase family and plays an essential role in T- and B-cell receptor signaling. The neoplastic cells in NLPHL, also known as popcorn cells or lymphocytic and histiocytic (LP) cells because of their distinctive morphology, express CD45. CD45 is typically absent of HRS in CHL. ^, CD45 has a strong membranous pattern of immunoreactivity. It is often difficult to discern CD45 expression in tumor cells owing to strong immunoreactivity of surrounding cells in CHL. One should look for areas without adjacent cells to determine if the tumor cells are CD45 immunoreactive.