Diagnostic Immunohistochemistry || Immunohistology of Hodgkin Lymphoma

137

• Introduction 137

• Biology of Antigens 137

• Antibody Specifications 144

• Diagnostic Immunohistochemistry 146

• Molecular Anatomic Pathology 150

• BeyondImmunohistochemistry:AnatomicMolecularDiagnosticApplications 150

• TheranosticApplications 150

• Summary 151

INTRODUCTIONHodgkin lymphoma (HL) is widely accepted to be a malignant clonal proliferation of B lymphocytes or, less often, T lymphocytes surrounded by variable numbers of inflammatory cells and fibrosis. The two major histo-logic types are classical Hodgkin lymphoma (CHL) and nodular lymphocyte predominance Hodgkin lymphoma (NLPHL). Classical Hodgkin lymphoma is further sub-divided into four subtypes:

• Lymphocyte rich type • Nodular sclerosis type • Mixed cellularity type • Lymphocyte depletion type (Figs. 5.1 and 5.2).1-3

NLPHL is a B-cell neoplasm derived from germinal center B cells that are continually undergoing somatic mutations of immunoglobulin genes.3-5 CHL belongs to the group of germinal center/post–germinal center B-cell lymphomas in which the Hodgkin/Reed- Sternberg cells (H/RSCs) have undergone extensive somatic muta-tions of immunoglobulin genes.6 Accordingly, these two major subtypes of HL express distinctive antigen profiles that can be used to distinguish NLPHL from the morphologically similar lymphocyte-rich variant of CHL. The German Hodgkin Study Group showed the

importance of using an immunohistochemical approach for improving the diagnostic accuracy of nodular lym-phocyte predominance Hodgkin lymphoma (NLPHL). Immunohistochemistry disproved the morphologic diag-nosis of NLPHL by an expert panel in 25 of 104 cases, whereas 13 cases originally not confirmed as NLPHL showed an NLPHD-like immunophenotypic pattern with a significantly better survival than CHL.7

BIOLOGY OF ANTIGENS

Principal Antibodies (CD45, CD20, CD30, CD15)The H/RSCs in NLPHL (also known as popcorn cells or lymphocytic and histiocytic (L&H) cells because of their distinctive morphology) generally express leuko-cyte common antigen (LCA) or CD45 (Fig. 5.3). In all cases of NLPHL and in a small number of CHL cases, there is an expression of B-cell antigen CD20 by the H/RSCs (Fig. 5.4).8-11 CD45 is usually absent or weakly expressed in a minor subset of H/RSCs in CHL (Fig. 5.5).12,13 In all cases of CHL and in a minor propor-tion of malignant cells in NLPHL, the tumor cells express CD30, a member of the tumor necrosis fac-tor superfamily (Fig. 5.6).14-16 Activation of CD30 signaling by native CD30L or Epstein-Barr virus latent membrane protein 1 (EBV-LMP1) results in activation of NF-кB transcription factor, which has an antiapoptotic effect, promotes cell proliferation, and causes up-regulation of cytokine production by H/RSCs.17,18 H/RSCs in CHL, but not NLPHL, express CD15 detected by antibody LeuM1 in 60% to 85% of cases, with an average of 68% (Fig. 5.7).19-22 The antigenic determinant for LeuM1 is a trisaccharide, 3-fucosyl-N-acetyllactosamine, which is formed by the 1-3 fucosylation of a type 2 blood group backbone chain (Gal1-4GlcNAc); the carbohydrate backbone is identical to that of Lewis X, also known as X-hapten.23

5ImmunohistologyofHodgkinLymphomaParul Bhargava • Marshall E. Kadin

IMMUNOHISTOLOGY OF HODGKIN LYMPHOMA138

GCCHetncNvCt(n

erminal Center/Post–Germinal enter Markers (BCL-6, CD138, D57)/RSCs in NLPHL, being germinal center derived,

xpress BCL-6, a transcription factor of germinal cen-er B cells, but they do not express CD10. They do ot express CD138/syndecan-1, a proteoglycan asso-iated with post–germinal center B cells.24 H/RSCs in LPHL often are surrounded by a population of acti-

ated helper-inducer memory T cells (CD4+, CD57+, D45R+, CD45+), which are normally confined to

he light zone of germinal centers of secondary follicles Fig. 5.8).4 Conversely, H/RSCs in CHL are heteroge-eous with respect to expression of BCL-6 and CD138,

reflecting their mixed germinal center or post–germinal center origin. H/RSCs in CHL typically do not display the T-cell rosetting characteristic of NLPHL.

TNF Superfamily (CD40)In addition to CD30, H/RSCs in CHL express CD40,25 an antigen that is characteristic of germinal center B cells and activation of which inhibits apoptosis (Fig. 5.9).26

Epithelial Membrane Antigen (EMA)EMA is expressed in some cases of NLPHL, but it is not expressed in CHL. EMA is however commonly ex pressed by tumor cells in anaplastic large cell lymphoma

A B

C D

EFIGURE 5.1 Classical Hodgkin lymphoma. (A) Lymphocyte rich. (B) Nodular sclerosis. (C) Lacunar cells in nodular sclerosis. (D) Mixed cellularity. (E) Lymphocyte depletion.

139BIOLOGY OF ANTIGENS

(ALCL), which may be useful as a distinguishing feature between ALCL and CHL.

T-Cell and Cytotoxic Markers (CD2, CD3, CD5, CD4, CD8, Granzyme B, Perforin, TIA-1)In 5% to 20% of CHL cases, H/RSCs appear to have vari-able expression of T-cell antigens (CD2, CD3, CD5, CD4, CD8) and antigens associated with cytotoxic molecules (granzyme B, perforin, and TIA-1) (Fig. 5.10).27-33 In one study, a mean fraction of 40% of H/RSCs (from 20% to

100%) expressed the analyzed T-cell markers.34 However, aberrant T-cell antigen expression was also detected in some CHLs with immunoglobulin gene rearrangements, presumably of B-cell derivation.35 A T-cell derivation was proven for H/RSCs by polymerase chain reaction amplifi-cation of T-cell receptor genes from single picked H/RSCs in approximately 1% to 2% of CHL cases.35-37

EBVEBV is associated with the etiology of HL, and EBV-LMP1 (or small RNAs of EBV known as EBERs) can be detected in about 50% of cases of CHL (Fig. 5.11).38

A B

FIGURE 5.2 Nodular lymphocyte predominance Hodgkin lymphoma. (A) Low magnification showing nodular pattern. (B) Popcorn vari-ants of Hodgkin/Reed-Sternberg cells.

A B

FIGURE 5.3 Expression of epithelial membrane antigen (EMA) in nodular lymphocyte predominance Hodgkin lymphoma. (A) Low magni-fication of nodule. (B) High magnification of individual Hodgkin/Reed-Sternberg cells.


A B

FIGURE 5.4 B-cell antigen expression in nodular lymphocyte predominance Hodgkin lymphoma. (A) Large Hodgkin/Reed-Sternberg cells in the center are surrounded by a nodule of smaller L26+ (CD20+) B lymphocytes. (B) CD20+ Hodgkin/Reed-Sternberg cells in lymphocyte predominance Hodgkin lymphoma.

FIGURE 5.5 Absence of CD45/LCA (leukocyte common antigen) on Hodgkin/Reed-Sternberg cells in classical Hodgkin lymphoma.

FIGURE 5.6 Hodgkin/Reed-Sternberg cells in classical Hodgkin lymphoma stained for CD30 with antibody Ber-H2.

FIGURE 5.7 Hodgkin/Reed-Sternberg cells in classical Hodgkin lymphoma expressing CD15 detected by antibody LeuM1.

FIGURE 5.8 Leu7+ (CD57+) T lymphocytes surrounding Hodgkin/Reed-Sternberg cells in nodular lymphocyte predominance Hodgkin lymphoma.


H/RSCs commonly express the EBV gene product latent membrane protein 1 (LMP-1), a transforming protein that can confer a growth advantage on H/RSCs.39,40 The frequency of EBV detection in HL is much higher in the mixed cellularity type and the lymphocyte depletion

type than in the nodular sclerosis type.40-42 EBV is fre-quently detected in CHL that occurs in immunocom-promised patients, such as those infected with human immunodeficiency virus (HIV) and those with post-transplant immunoproliferative disorders.43 EBV is also detected at higher frequency in HL patients in develop-ing countries.41,42

Dendritic or Antigen Presenting Cell Markers: FascinFascin is a relatively new sensitive marker that has been described for H/RSCs in CHL.44 Fascin is a 55-kD actin-bundling protein that is localized predominantly in den-dritic cells in non-neoplastic tissues. The staining profile for fascin raises the possibility of a dendritic cell deriva-tion (particularly an interdigitating reticulum cell) for the neoplastic cells of HL, notably in nodular sclerosis (Fig. 5.12). However, because fascin expression can be induced by EBV infection of B cells, the possibility of viral induction of fascin in lymphoid or other cell types must also be considered.44 While fascin expression has been shown in all cases of CHL, giving it a high

FIGURE 5.9 Hodgkin/Reed-Sternberg cells in CHL expressing CD40.

A B

FIGURE 5.10 CHL with T-cell phenotype. (A) Hodgkin/Reed-Sternberg cells stained for UCHL1 (CD45RO). (B) Expression of cytotoxic molecule TIA-1 by Hodgkin/Reed-Sternberg cells and smaller surrounding tumor-infiltrating lymphocytes.

A B

FIGURE 5.11 Detection of Epstein-Barr virus in classical Hodgkin lymphoma. (A) Expression of latent membrane protein-1 (LMP-1). (B) Insitu hybridization studies for EBV encoded small RNAs known as EBER.


negative predictive value, it is not specific for CHL. Fas-cin expression has also been described in a majority of cases of ALCL (50% to 70%),45,46 which makes it less useful for this differential.

CLIP-170/RestinH/RSCs of CHL strongly express CLIP-170/restin, which colocalizes with membranes of intermediate mac-ropinocytic vesicles, assisting in the trafficking of mac-ropinosomes to the cytoskeleton. This is a crucial step in antigen presentation. The strong expression of CLIP-170 restin in H/RSCs, dendritic cells, and activated B cells underscores their functional similarities, support-ing a function-based concept of H/RSCs as professional antigen-presenting cells.47

B-Cell Markers (CD79a) and Transcription Factors (BSAP, Oct-2, BOB.1, JunB)CD79 is a dimeric, transmembrane protein, which, along with surface immunoglobulin, is part of the B-cell receptor complex.48 It is a pan B-cell marker expressed from the pre-B stage to the plasma cell stage of differen-tiation.49 Like CD20, CD79 is generally expressed in all cases of NLPHL. However, H/RSCs of CHL are generally non-immunoreactive for CD79; in a minor subset (0% to 20%) of cases, a small proportion of the neoplastic cells may be positive.11,50-52 Global loss of B-cell–specific gene expression is a distinctive feature of H/RSCs in CHL.53 The loss may be due to aberrant expression of ID2, a suppressor of B-cell–specific gene expression in HL.54

B-cell–specific activator protein (BSAP) is a tran-scription factor expressed in B cells and B-cell–derived lymphomas. It is encoded by the PAX-5 gene and influences several B-cell functions such as B-cell anti-gen expression, Ig expression, and class switch. It is expressed in the majority of H/RSCs in CHL55 as well as L&H cells of NLPHL,56 further supporting their

B-cell origin. In contrast, BSAP is not expressed in nor-mal or malignant T cells, and thus is absent in T/null cell ALCLs.55

Oct-2 is a transcription factor, which, along with its co-activator BOB.1/OBF.1, binds to immunoglobulin gene octomer sites, thus inducing immunoglobulin synthesis.57 Germinal center B cells normally demon-strate strong staining for Oct-2 and BOB.1. Because they are germinal center derived, L&H cells in NLPHL are consistently immunoreactive for both markers.56 Conversely, the H/RSCs in CHL do not express both (80%) or express only one (20%) of the two proteins.58,59 H/RSCs often do not express immunoglobulin (Ig), which is thought to be due to crippling mutations within Ig genes; absence of transcriptional activators such as Oct-2/BOB.1 may represent novel mecha-nisms for Ig dysregulation.58 Although most T-cell lymphomas are Oct-2–negative, variable staining has been demonstrated in some peripheral T-cell lympho-mas not otherwise specified (NOS) as well as a subset (~50%) of anaplastic lymphoma kinase (ALK)-positive ALCLs.60

JunB and c-Jun are part of the Activator Protein-1 (AP-1) family of transcription factors. AP-1 proteins are stimulated in a rapid and transient fashion by a number of extracellular signals that trigger growth factor pathways and/or stress signals (e.g., UV radia-tion). They promote mitogen-induced cell-cycle pro-gression as well as regulate apoptosis. Recently it has been demonstrated that H/RSCs in CHL constitutively overexpress AP-1 proteins containing c-Jun and JunB (Fig. 5.13). Conversely, malignant cells in NLPHL had been shown to express neither c-Jun nor JunB.61 How-ever, in our experience,60a JunB is expressed in a minor subset of NLPHL cases. Additionally, JunB antibody stains scattered lymphocytes, particularly in areas of progressively transformed germinal centers. Most of the other B- and T-cell NHLs tested did not express or only weakly expressed JunB and/or c-Jun, with the exception of t(2;5) positive ALCLs (which showed strong expression).61

A B

FIGURE 5.12 Fascin expression in nodular sclerosis HL. (A) Low magnification of nodule. (B) Staining of individual Hodgkin/Reed- Sternberg cells at high magnification.


Immunoglobulins (J Chain, IgD) J-chainImmunoglobulins (J chain, IgD) J-chain is a 15-kD acidic protein synthesized by B cells and plasma cells that secrete polymeric immunoglobulins. J-chain expression has thus been observed in most H/RSCs in NLPHL, but not in H/RSCs of CHL with dysregulated Ig genes.58,62,63 IgD expression in H/RSCs has been reported in a sub-set (27%)64 of NLPHL cases with an extra-follicular distribution of L&H cells and a relatively T-cell–rich background. In contrast, IgD expression is rarely seen in T-cell–rich B-cell lymphoma (TCRBCL). Some stud-ies demonstrated immunoreactivity for IgD in a minor subset of H/RSCs in CHL65; however, others were negative.66

CD74CD74 functions as an MHC class II chaperone and is normally expressed by a variety of cell types including B cells, activated T cells, macrophages, activated endo-thelial cells, and epithelial cells. Although expressed by the H/RSCs of CHL, CD74 is not specific for this lymphoma; expression has been reported in a variety of non-Hodgkin lymphomas as well as in non-lymphoid epithelial malignancies.

Other Biological Markers in Hodgkin LymphomaIt has been shown that expression of translation ini-tiation factors eIF-4E and eIF-2alpha is increased in neoplastic cells of Hodgkin lymphoma, but not in sur-rounding lymphocytes.67 An increase in eIF-4E ex pression may lead to constitutively high expression of NF-кB. H/RSCs have high expression of c-FLIP, which pro-tects cells from apoptosis.68 Tissue inhibitor of metal-loproteinases (TIMP-1 and TIMP-2) are proteins with proteinase inhibition and cytokine properties. TIMP-1

is active primarily in B cells and B-cell lymphomas, whereas TIMP-2 is restricted to T cells. HL-derived cell lines express TIMP-1, with low expression of TIMP-2. TIMP-1 protein can be detected in frozen tissues of CHL lymph nodes, where it produces primarily a dif-fuse background staining and co-localization with CD30 in few H/RSCs.69 Galectin-1 is an immunoreg-ulatory glycan-finding protein that is expressed by H/RSCs. H/RS cell Gal-1 may contribute to the develop-ment and maintenance of an immunosuppressive Th2/Treg-skewed microenvironment in CHL and provide the molecular basis of selective Gal-1 expression in H/RSCs.70 Programmed death-1 (PD-1) ligand (PD-L) sig-naling system is involved in the functional impairment of T cells such as in chronic viral infection or tumor immune evasion. PD-L expression is up-regulated in H/RSCs in tissues and cell lines as well as some T-cell lym-phomas but not in B-cell lymphomas. PD-1 is elevated markedly in tumor-infiltrating T cells of HL and in peripheral blood T cells of HL patients.71

CYTOKINESINHODGKINLYMPHOMA

Clinical and histologic features of Hodgkin lymphoma have been associated with cytokine and chemokine pro-duction by tumor cells.72,73 The majority of CHL cases are characterized by expression of tumor necrosis fac-tor receptor (TNFR) family members and their ligands and an unbalanced production of Th2 cytokines and chemokines.74 Chemokine receptor CCR7 is a lympho-cyte homing receptor expressed in B, T, and activated dendritic cells and has been implicated in regulation of lymphocyte migration to secondary lymphoid organs. The promoter region of CCR7 has binding sites for both AP-1 and NF-кB. In line with the c-Jun/JunB over-expression seen in CHL but not in NLPHL, CCR7 is over-expressed in most H/RSCs of CHL (Fig. 5.14).75 CD25 H/RSCs in CHL manifest variable expression of CD25 (Tac, p55), the alpha unit of the receptor for interleukin-2 (IL-2).76,77 CD25 is not expressed by H/RSCs in NLPHL. Aggregation of TRAF adapter

FIGURE 5.13 JunB expression in classical Hodgkin lymphoma. FIGURE 5.14 Hodgkin/Reed-Sternberg cells in classical Hodgkin lymphoma expressing CCR7.


proteins TRAF2 and TRAF5 in H/RSCs is required for CD30 signaling and activation of NF-кB.78 TGF-beta and basic fibroblast growth factor produced by HL cells are associated with the pathogenesis of nodular sclerosis.79,80 IL-13 and IL-13 receptors are frequently expressed in H/RSCs and contribute to the production of TGF-beta 1– mediated fibrosis.81,82 H/RSCs secrete IL-5, which stimulates production of eosinophils and eosinophilia.83 H/RSCs also express eotaxin, which is a chemo-attractant for eosinophils.84 Both neoplastic and reactive IL-10–producing cells are significantly more common in EBV+ HL cases. IL-10 is an immunosup-pressive cytokine and can help H/RSCs to escape local immune surveillance.85 STAT proteins (STATs) are a family of transcription factors responsible for signal transducers and activators of transcription. STAT 3, STAT5, and STAT6 are frequently constitutively acti-vated in H/RSCs and can be demonstrated by immu-nohistochemistry. STAT5 appears to be activated by IL-21.86 STAT 6 mediates signaling by IL-13, and antibody-mediated neutralization of IL-13 causes sig-nificant decreases in levels of HL cell proliferation and phosphorylated STAT6 in HL cell lines.87

Table 5.1 summarizes the expression pattern of some of the new biological markers in HL as well as other entities in the differential diagnosis of HL.

ANTIBODY SPECIFICATIONSThe antibodies most commonly used for diagnosing HL are Ber-H2 (CD30), LeuM1 (CD15), LCA (CD45), L26 (CD20), CD75 (LN1), CD74 (LN2), PAX5, CD3, UCHL1(CD45RO), ALK-1, fascin, and EBV-LMP1. EMA and CD57 can be used to recognize NLPHL. Monoclonal antibody LN1 reacts with H/RSCs in about one third of HL cases, most frequently in cases of NLPHD (>75% of cases).10 Monoclonal antibody LN2, which recognizes the MHC class II-associated invariant chain, reacts with H/RSCs in approximately two thirds of HL cases.10 All the antibodies mentioned previously can be used in formalin-fixed paraffin-embedded tissues. Additional antibodies that are useful

in the diagnosis of difficult cases are BNH.988 and CBF.78 (Fig. 5.15 and Table 5.2).89

For antigen retrieval, we have replaced the use of a microwave oven with a steamer that heats the sections to 95°C to 98°C. The slides are immersed in Coplin jars containing citrate buffer (pH 6, 0.01 mol/L) and heated in the steamer for 20 minutes. Afterward, slides are cooled at room temperature for 30 minutes, rinsed in double-distilled water, and then transferred to phos-phate-buffered saline, pH 7.4.

Immunostaining PitfallsCD15ANTIGEN

When one relies on the demonstration of CD15 to make a diagnosis of HL, problems can arise because more than 30% of CHL cases will not express CD15 detected by LeuM1 antibody. A comparative study by Ree and coworkers, which we confirmed in our laboratory, showed that anti-Lewis-X (BG-7) (Signet Laboratories, Dedham, MA) is superior to LeuM1 for staining H/RSCs, yielding 87% versus 68.5% for LeuM1 (Fig. 5.16).90 It is also important to identify the cells that express CD15 because granulocytes express high levels of CD1519 and are often present in various tumors other than HL.

CD30ANTIGEN

The use of monoclonal antibody Ber-H2 together with antigen retrieval methods has enabled sensitive detec-tion of CD30 in formalin-fixed paraffin-embedded tissues. However, some hematopathologists prefer to use B5-fixed tissues, which afford excellent cytomorphology of lymphoid tissues. B5 is a mercuric chloride–containing

TABLE 5.1 NewBiologicalMarkersofHodgkin/Reed-SternbergCells

Antigen CHL NLPHL TCRBCL ALCL

NF-кB + U U* –

JunB/c-Jun + S U +

CCR7 + – U U

Oct-2/BOB.1 S** + + S

J-chain – S S –

BSAP/PAX5 + (weak) + + –

+, Nearly all cases positive; S, sometimes positive; R, rare (<5%); –, negative; U, expression unknown.

*+/– in diffuse large B-cell lymphoma (DLBCL); not directly studied in TCRBCL.

**Both (80%); one (20%).

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

CD30 CD45 ALK-1

100%

HL classicNLPHDALCL

FIGURE 5.15 Frequency of antigens in classical Hodgkin lympho-ma, nodular lymphocyte predominance Hodgkin lymphoma, and anaplastic large cell lymphoma.

145ANTIBODY SPECIFICATIONS

fixative that requires removal of mercury before immunostaining. This is usually accomplished with Lugol’s solution followed by sodium thiosulfate. A study by Facchetti and coworkers showed that omit-ting the Lugol’s treatment is optimal for detection of CD30, even without wet heating with microwave or proteolytic predigestion of sections.91

CD45(LCA)

It is often difficult to determine CD45 expression in tumor cells owing to strong immunoreactivity of sur-rounding cells in CHL. One should look for areas with-out adjacent cells to determine if the tumor cells are CD45 immunoreactive.

TABLE 5.2 AntibodiesforDetectionofHodgkinLymphoma–AssociatedAntigens

Antibody Clone Manufacturer Dilution TypeofAntigen-RetrievalMethod

CD30 Ber-H2 Dako 1:25 Steamer/citrate buffer pH 6, 20 min at 95°C to 98°C

CD15 LeuM1 Becton-Dickinson 1:25 Same as CD30

CD45 (LCA) 2B11 Dako 1:200 Same as CD30

CD20 L26 Dako 1:100 Same as CD30

CD45RO UCHL1 Dako Pepsin digest 10 min at 37°C

CD3 UCHT1 Dako Steamer/citrate buffer pH 6, 20 min at 95°C to 98°C

CD40 MAB89 Immunotech 1:40 Same as CD3

ALK-1 ALK-1 Dako 1:25 Same as CD3

Fascin 55K-2 Dako 1:75 Same as CD3

Lewis-X type 2 chain (BG-7)

P12 Signet 1:40 Steamer/citrate buffer pH 8, 20 min at 95°C to 98°C

EBV-LMP1 CS1-4 Dako 1:50 Steamer/citrate buffer pH 6, 20 min at 95°C to 98°C

BCL-6 PG-B6p Dako 1:10 Same as EBV-LMP1

CD57 Leu7 Dako 1:10 Same as EBV-LMP1

EMA E29 Dako 1:50 Pepsin digest, 12 min at 37°C

CDw75 LN1 ICN Biomedicals Undiluted Steamer/citrate buffer pH 6, 20 min at 95°C to 98°C

CD74 LN2 ICN Biomedicals Undiluted Same as CDw75

NF-кB P65C Zymed Lab 1:200 Same as CDw75

CCR7 CCR7.6B3 eBioscience, San Diego, CA

1:200 Steam for 30 min in 1 mM EDTA pH 8.089a

JunB SC8051 Dako 1:75 HIER steamer/Target Retrieval Solution 30 min

A B

FIGURE 5.16 Comparative staining of Hodgkin/Reed-Sternberg cells for CD15 antigenic determinant with (A) anti-Lewis-X and (B) anti-LeuM1 antibodies in a case of lymphocyte depletion classical Hodgkin lymphoma shows increased sensitivity with anti-Lewis-X antibody.


CD20ANDT-CELLANTIGENS

With CD20 and T-cell antigen staining in CHL, similar difficulties can arise when interpreting the staining of tumor cells versus its surrounding cells.

DIAGNOSTIC IMMUNOHISTOCHEMISTRYAlthough CHL and NLPHL have distinctive mor-phologic and immunophenotypic characteristics, it is important to note that an inconsistency of immu-nophenotype of H/RSCs has been reported in simul-taneous and consecutive specimens from the same patients in paraffin sections. Chu and coworkers found that the immunophenotype of H/RSCs was identi-cal in simultaneous biopsies in only 11 of 39 (28%) patients and remained constant in consecutive biopsies in only 4 of 21 (19%) patients.92 Major differences were related to cell lineage–specific antigens, whereas minor differences involved mainly CD15 and CD74 antigens.

In most cases of CHL and NLPHL, an accurate diagnosis can be rendered with an adequate size biopsy, proper fixation, thorough morphologic review, and appropriate antibody selection. However, there are several caveats and differential diagnostic consid-erations that may cause confusion with HL. One of the major differential diagnoses includes non- Hodgkin lymphoma subtypes with an abundance of non- neoplastic reactive background inflammatory cells. We will now discuss these subtypes, as well as certain non-lymphoid malignancies and tumor-like non-neoplastic look-alikes.

Non-Hodgkin LymphomasANAPLASTICLARGECELLLYMPHOMA

CD30 is displayed on the tumor cells of ALCL, which is a non-HL with a different natural history than HL.14,93-96 The histologic features of ALCL, particularly cohesive growth pattern and lymph node sinus infiltration by tumor cells, were thought to be distinguishing char-acteristics of ALCL; however, experience has shown otherwise. Rare cases of cell-rich HL—particularly those classified as nodular sclerosis type II in the Brit-ish National Investigation,97 or the syncytial vari-ant98—and some cases of lymphocyte-depletion HL can be confused with ALCL (Fig. 5.17).99 In these cases, a panel of antibodies is used to make the dis-tinction (Tables 5-1 and 5-3). Perhaps most useful is the monoclonal antibody ALK-1 directed against the ALK tyrosine kinase, which is most often activated by the translocation t(2;5)(p23; q35) and less frequently by other chromosomal rearrangements in ALCL (Fig. 5.18).100,101 ALK is rarely, if ever, expressed in the malignant cells of HL.99 Demonstration of a B-cell phenotype (as with subset CD20 expression or BSAP/PAX5) in H/RSCs in CHL is also useful in excluding T-ALCL.

LYMPHOEPITHELIOIDCELLVARIANTOFPERIPHERALT-CELLLYMPHOMA(LENNERTLYMPHOMA)

Lennert lymphoma is another T-cell lymphoma that can resemble CHL because of the presence of H/RS-like cells, eosinophils, and plasma cells. Small clusters of epi-thelioid histiocytes resembling granulomas are a distinc-tive feature. In Lennert lymphoma, the H/RS-like cells express a CD4+ T-cell phenotype.102

PRIMARYMEDIASTINALB-CELLLYMPHOMA

Primary mediastinal B-cell lymphoma (PMBCL) can be confused with HL because it presents as a mass in the anterior mediastinum of young adults and often contains H/RS-like cells in a background of collagen sclerosis (Fig. 5.19).103 Also, in one study, H/RS-like cells expressed CD30 in 35 of 51 (69%) cases.104 However, PMBCL can be distinguished from HL by the strong, uniform expres-sion of CD20, lack of CD15, absence of EBV (EBERs and LMP-1), and lack of inflammatory background, par-ticularly eosinophils (which is characteristic of HL).

T-CELL–RICHB-CELLLYMPHOMA

T-cell–rich B-cell lymphoma was recognized as a non-Hodgkin lymphoma (NHL) usually occurring in patients older than 50 years with advanced (stage III or IV) disease. Response to commonly used chemotherapy regimens for HL is poor. Therefore, it is important to distinguish TCRBCL from HL, particularly NLPHD or LRCHL (Fig. 5.20).104,105 The tumor cells in TCRBCL appear to be negative for CD30 and CD15, as well as for vimentin, all of which are expressed in H/RSCs in CHL. Furthermore, the reactive inflammatory infiltrate that is rich in TIA-1+ lymphocytes in TCRBCL and CHL is rarely encountered in NLPHD, whereas CD57+ lymphocytes characteristic of NLPHD are infrequent in TCRBCL.106 NLPHL gener-ally has at least one nodular area; the background cells in the nodules of NLPHL are B-cell rich in contrast to the T-cell–rich background of TCRBCL.

HODGKIN-LIKEPOST-TRANSPLANTLYMPHOPROLIFERATIVEDISORDER(HL-PTLD)

HL-PTLD, with cells resembling H/RSCs (Fig. 5.21), has been reported in allograft recipients, post- methotrexate therapy patients, 107 and HIV-infected patients. CHL has also been reported in each of these conditions, and differential diagnosis is based on morphologic and immunophenotypic features.108 The histopathologic features often show a mixed population of small- to intermediate-sized lymphocytes admixed with histio-cytes, plasma cells, and rare eosinophils and neutrophils, as well as scattered large pleomorphic mononuclear and binucelated cells without sclerosis and no nodularity, resembling mixed cellularity or lymphocyte depletion Hodgkin lymphoma.109 While H/RSCs in CHL char-acteristically express CD30 and CD15, HL-PTLD cells often have an activated B-cell phenotype (i.e., CD20+, CD30+, CD45+, but CD15–). The atypical cells in

147DIAGNOSTIC IMMUNOHISTOCHEMISTRY

HL-PTLD have been reported to express fascin, with a weak expression of BCL-2; CD45 is variably expressed. Virtually all cases of HL-PTLD are EBV-positive.

CHLINCLLANDHODGKIN-LIKECELLSINCLL

Classical HL transformation is a rare form of Richter’s transformation, which can occur in patients with B-chronic lymphocytic leukemia (B-CLL). H/RSCs are seen in a polymorphous background of inflammatory

cells, and are morphologically and immunophenotypi-cally indistinguishable from CHL. Such transformations have been variously reported to be clonally distinct110-112 or clonally related112,113 to B-CLL. Separately, H/RS-like cells may be seen (singly or clustered) in a background of B-CLL. Although RS-like cells in CLL are typically CD30-immunoreactive, with variable expression of CD20, CD15, and LMP1, the background is composed of monomorphous B-CLL cells and thus these cases are not thought to represent Richter’s transformation.

A B

C D

E F

FIGURE 5.17 Cell-rich classical Hodgkin lymphoma with interfollicular and intrasinus distribution of tumor cells. (A) Low magnification of interfollicular pattern. (B) Low magnification of sinus infiltration. (C) High magnification of Hodgkin/Reed-Sternberg cells within sinus of lymph node. (D) Expression of CD30 by Hodgkin/Reed-Sternberg cells within sinus. (E) Expression of fascin by Hodgkin/Reed-Sternberg cells. (F) Expression of CD40 by Hodgkin/Reed-Sternberg cells within sinus.


NCism

Fw

+

onlymphoid TumorsD30, the most consistent marker of H/RSCs, is read-

ly detected in formalin-fixed, paraffin-embedded tis-ues.14,114 However, tumor cells in some nonlymphoid alignancies, including embryonic carcinoma, melanoma,

and pancreatic cancer, can also express CD30.114,115 Because sinus infiltration of lymph nodes is charac-teristic of CD30+ ALCLs, there is potential for confu-sion of ALCLs with the few metastatic carcinomas that express CD30 antigen.14 Moreover, because malignant

A B

C D

IGURE 5.18 Hodgkin-like anaplastic large cell lymphoma with (A) nodular pattern and (B) lacunar cells. (C) CD30 staining of tumor cells ithin sinus. (D) Expression of p80NPM/ALK by tumor cells in lacunar spaces.

TABLE 5.3 AntibodyPanelforDifferentialDiagnosisofHodgkinLymphoma

LymphomaType

ClassicalHodgkinLymphoma

NodularLymphocytePredominanceHodgkinLymphoma

AnaplasticLargeCellLymphoma

PrimaryMediastinalLargeB-CellLymphoma

T-Cell–RichB-CellLymphoma

CD30 + S + S –

CD15 + – R – –

CD20 S + – + +

CD3 – – + – –

CD40 + + – + +

CD45 – + S + +

EBV-LMP-1 S – – – –

ALK – – + – –

Fascin + – – – –

, Nearly all cases positive; S, sometimes positive; R, rare (<5%); –, negative.

149DIAGNOSTIC IMMUNOHISTOCHEMISTRY

melanoma can express CD30, there is the possibility of mistaking an anaplastic melanoma for a primary CD30+ ALCL.115

CD15 expressed on H/RSCs is also associated with carcinomas.116 Fortunately it is not common to encoun-ter a carcinoma that could be clinically or histologically mistaken for HL. However, the cohesive growth pattern of tumor cells in the syncytial variant of nodular scle-rosis HL might rarely be mistaken for metastatic carci-noma expressing CD15.

Pseudoneoplastic Look-alikesINFECTIOUSMONONUCLEOSIS

H/RS-like cells in infectious mononucleosis are similar in most respects to their morphologic counterparts in HL with respect to expression of EBERs, EBV-LMP1, and CD30, and low expression of CD45.117 However, the H/RS-like cells in infectious mononucleosis are CD15–.118

CYTOMEGALOVIRUSLYMPHADENITIS

Lymph nodes infected with cytomegalovirus contain H/RS-like cells that are caused by viral inclusions and are readily distinguished from HL by absence of CD15 and CD30 (Fig. 5.22).

INTERFOLLICULARLYMPHADENITIS

Lymphadenitis mimicking Hodgkin disease has been described as a benign lymphadenopathy that can mimic interfollicular HL.119,120 Cervical lymph nodes are affected most often. There is no progression to lym-phoma. The lymph nodes show follicular hyperplasia with a mottled interfollicular zone with epithelioid histiocytes, lymphocytes, eosinophils, and immuno-blasts. Some immunoblasts with prominent nucleoli resemble H/RSCs. However, their nucleoli are typically smaller and basophilic, in contrast to the eosinophilic nucleoli of H/RSCs. Immunohistochemistry distin-guishes this disorder from interfollicular HL because

FIGURE 5.19 Hodgkin/Reed-Sternberg–like cells in primary medi-astinal B-cell large cell lymphoma.

FIGURE 5.20 Hodgkin/Reed-Sternberg–like cells in T-cell–rich B-cell lymphoma.

FIGURE 5.21 Hodgkin-like post-transplant lymphoproliferative disorder.

FIGURE 5.22 Hodgkin/Reed-Sternberg–like cells in lymph node infected by cytomegalovirus.


the H/RS-like cells display B- or T-cell antigens and lack CD15.120,121

GRANULOMATOUSLYMPHADENITIS

The presence of non-caseating granulomas is a well-known histologic feature associated with several non- hematopoietic and hematopoietic malignancies, including HL (Fig 5.23). In HL, approximately 15% of patients have granulomas, which may be present in nodal and extranodal sites uninvolved by HL;122 the presence of granulomas alone, in the absence of diagnostic H/RSCs, should not be interpreted as evidence of HL. Conversely, granulomatous reaction may be present in a site involved by HL and on occasion may be extremely florid, necessitating a thorough morphologic review to detect small foci of HL. H/RSCs in such areas have the classical immunophenotype (CD30+, CD15+, LCA–, CD20–) as opposed to reactive immunoblasts (LCA+, CD20+, CD30+, CD15–).

MOLECULAR ANATOMIC PATHOLOGYIn a majority of cases, the diagnosis of CHL and NLPHL is based on morphology and immunophenotyping, and molecular testing is rarely utilized. Polymerase chain reaction studies (PCR) for immunoglobulin heavy chain gene (IgH) rearrangements have been used to distin-guish T-cell–rich large B-cell lymphoma from HL, with the former demonstrating clonal bands more frequently than the latter.123 While demonstration of clonal IgH bands in CHL generally required special cell enrichment techniques and/or single cell microdissection, recent data suggest that detectable rearrangements without microdissection can be seen in a higher number of CHL cases using newer multiplex primers, making IgH analy-sis less useful in this context.124

Most T-HL (and a subset of B-HL) have TCR gam-ma rearrangements.35 In one case of CHL, identical

rearrangements of the TCR alpha chain were found in the HL lymph node and co-existent cutaneous T cell lym-phoma.125 In another case studied by single-cell PCR, identical TCR beta chain rearrangements were found in H/RSCs in a lymph node of mixed cellularity HL and skin lesions with CD30+ CD15+ H/RSCs.37

BEYOND IMMUNOHISTOCHEMISTRY: ANATOMIC MOLECULAR DIAGNOSTIC APPLICATIONSThe exact genomic alteration that drives lymphomagen-esis in CHL and NLPHL is not completely understood. Nuclear factor of kappa light polypeptide gene enhancer in B cells 2 (NF-кB) is present in numerous cell types and is normally only transiently activated by stress, immune, and inflammatory signals. It has been shown that NF-кB is constitutively activated in cultured H/RSCs,17 primar-ily owing to mutations and/or increased turnover of its natural inhibitor I-kappa B (IкB).74 Constitutive NF-кB results in overexpressed anti-apoptotic genes that allow H/RSCs to escape apoptosis despite losing capability to produce Ig. Overexpression of NF-кB can be demon-strated immunohistochemically in CHL; however, it is not specific to this tumor type and can be seen in a vari-ety of other malignancies, including mediastinal B-cell lymphoma.126,127

A tissue microarray study using immunohistochem-istry and in situ hybridization to observe cell cycle and apoptosis regulating genes has shown multiple altera-tions in cell cycle checkpoints and major tumor suppres-sor pathways, some of which are linked to survival as well as EBV positivity.128

THERANOSTIC APPLICATIONSAlthough newer therapeutic agents such as monoclonal antibodies are being evaluated, most are in early experi-mental stages. Nonetheless, accurate documentation of H/RSC expression of potential therapeutic targets would likely be beneficial when evaluating such biopsies.

CD20 and Monoclonal Antibody (Rituximab) TherapyCD20 expression by a majority of L&H cells in NLPHL has been used as a basis for targeted monoclonal ther-apy.129 Rituximab therapy is also being tried in CHL, not only targeting the minor CD20 expressing H/RSCs but also infiltrating background B-lymphocytes.130

CD40CD40 is widely expressed on H/RSCs, and its ligand, CD40L, is expressed by many T cells surrounding H/RSCs. In B cells, CD40 regulates progression from immu-noglobulin isotype switch to cytokine secretion and ultimately terminates in Fas-mediated apoptosis to ter-minate the immune response. CD40 signal transduc-tion pathways result in activation of NF-кB; this in turn

FIGURE 5.23 Granulomas in a lymph node involved by Hodgkin lymphoma.

151SUMMARY

activates transcription of IL-2, IL-6, IL-8, TNF, and GM-CSF, thus affecting the proliferation and activation of many components of the immune system. CD40 acti-vation of NF-кB is mediated by proteolysis of TRAF3, and a protease inhibitor has been used to block this pathway.131

CD30Anti-CD30 monoclonal antibodies have been used in pre-clinical murine xenograft models of localized HL to demonstrate dose-dependent reduction in tumor mass. There is also a significant increase in survival of mice bearing disseminated HL treated with anti-CD30.132 Anti-tumor activity of anti-CD30 has been enhanced by conjugation with monomethyl auristatin E, which induces G2/M growth arrest and cell death.133 Trials using monoclonal anti-CD30 antibodies as well as bi-specific molecules are also being conducted.134,135

Anti-EBV TherapyIn almost 40% of HL patients, H/RSCs express EBV-associated antigens. EBV-specific cytotoxic T lym-phocytes expressing the anti-CD30ζ artificial chimeric T-cell receptor have been employed for immunotherapy of HL. Adoptive transfer of EBV-specific cytotoxic T lymphocytes (EBV-CTLs) have shown that these cells persist in patients with HL to produce complete tumor responses.136 Treatment failure occurs if a subpopu-lation of malignant cells lacks or loses expression of EBV antigens. To overcome this limitation, investiga-tors have prepared EBV-CTLs that retained antitumor

activity conferred by their native receptor while express-ing a chimeric antigen receptor specific for CD30.137

Interleukin-2 ReceptorInterleukin-2 receptor (CD25) has been used as a target for immunotherapy protocols to treat HL.138 A pitfall of these protocols is that difficulty is sometimes encoun-tered in demonstrating CD25 expression by H/RSCs against which the therapy is directed. Indeed, CD25 is often expressed on activated tumor-infiltrating lympho-cytes (TILs) in HL, and it is important to distinguish them from H/RSCs. We found this possible in most cases when a biotinylated tyramine enhancement step139 was applied to formalin-fixed paraffin-embedded tissues.77

CCR4CCR4 is a chemokine receptor expressed on H/RSCs in 24% of patients with HL. A chimeric anti-CCR4 anti-body KM2760, the Fc region of which is defucosylated to enhance antibody dependent cellular cytotoxicity, is being developed as a novel treatment for patients with CCR4+ HL.140

SUMMARYThe diagnosis of HL from routine H&E sections is often readily made. However, increasing recognition of new lymphoma types with overlapping morpholo-gies dictates the use of immunohistochemistry to avoid incorrect diagnoses. Among the non-Hodgkin lymphomas that may be confused with HL are ALCL

CD30+CD15–

CD45–

CD20+ CD20– Keratin+

Carcinoma

CD43, T-antigens: any+Alk-1+(–)

CD43, T-antigens: all –Alk-1–

Classical HL PAX5/BSAP– PAX5/BSAP+

Classical HL

CD30+CD15+

CD20–/+, PAX5+Fascin+

CD20–, PAX5–T-antigens+

CHL

PTCL

Atypical T-cellbackground

T-cell clonality

CHL (1-2%)

Classical HL

Keratin–

Sarcoma (includinggranulocytic sarcoma)ALCLKeratin+ or

S-100+

Embryonal CA;pancreatic CA;

malignant melanoma

Keratin–S-100–

CD30–CD15+/–

FIGURE 5.24 Diagnostic algorithm: Classical Hodgkin lymphoma.


and T-cell–rich B-cell lymphoma. It is also important to distinguish NLPHL from lymphocyte-rich CHL by aid of immunohistochemistry. At the same time, one must be aware of antigens shared by H/RSCs and NHL cells. Recognition of certain antigens expressed or not expressed by H/RSCs informs us of the biology of these cells. The silencing of B-cell antigens by Id2 and possibly other mechanisms makes the diagnosis of HL difficult in some cases; however, it distinguishes H/RSCs from B cells in most NHLs. Finally, one must always be cognizant of pseudoneoplastic conditions that mimic HL, wherein immunohistochemistry is a useful aid to arrive at the correct diagnosis. Overall, immunohistochemistry contributes significantly to our knowledge of HL as well as better patient management. One possible immunohistologic approach to resolve differential diagnostic considerations is presented in the supplied diagnostic algorithm (Figs. 5.24, 5.25).

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CD30+CD15–

LMP+or EBER+

Primarymediastinallymphoma

DLBCL(anaplastic)

CD30+

Infectiousmononucleosis NLPHL TCRLBCL

H/O transplant;methotrexate

HIV

PTLD

Anaplasticmorphology

MediastinalNo inflammatory

backgroundALCL

B-cell nodulesCD57 rosetting

EMA+

BackgroundT-cells TIA-1+

CD30–CD15–

CD45

CD20+CD20–T-cell markers+

Alk-1 ±

CD20+T-cell markers–

FIGURE 5.25 Diagnostic algorithm: B-cell lymphomas, NLPHL, and pseudoneoplastic lymphoproliferative disorders.

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Diagnostic Immunohistochemistry || Immunohistology of Hodgkin Lymphoma

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