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The Pathology of Hodgkins Disease


PATRICK A. TRESELER, MD, PHD

HISTORY In 1832, Dr. Thomas Hodgkin, working at Guys Hospital in London, reported a series of seven cases of enlargement of the lymph nodes and spleen, which he speculated might represent a primary disorder of those tissues rather than reactive enlargement.1 Hodgkins observations on these cases may well have slipped into permanent obscurity had they not been resurrected some 30 years later by another Guys Hospital physician, Dr. Samuel Wilks, who, in a paper published in 1865, was the first to attach the name Hodgkins disease to this clinical entity.2 The first descriptions of the microscopic appearance of the affected tissues appeared in the late 1800s.3 However, it was not until the seminal observations by Sternberg in 18984 and Reed in 19025 that widespread attention was drawn to the curious giant cell present in many such cases, which would later come to bear their names and which would soon become regarded as a morphologic hallmark of Hodgkins disease (HD) (Figure 201). Interestingly, neither Sternberg nor Reed felt that this disorder represented a neoplasm. Sternberg believed that it was a variant of tuberculosis,4 whereas Reed felt it to be an independent entity of inflammatory origin.5 The work by Sternberg and Reed gave the world its first pathologic definition of HD: the identification of diagnostic Reed-Sternberg (RS) cells in an appropriate cellular environment. This essentially morphologic definition of HD proved useful in separating cases of HD from other inflammatory and neoplastic conditions that might mimic HD clinically and provided the basis for the diagnosis of HD for most of the twentieth century.68 In this definition, a

diagnostic RS cell was defined as a giant cell with multiple (frequently two) nuclear lobes, each containing a prominent inclusion-like acidophilic nucleolus that itself approached the size of a small lymphocyte (Figure 202). Mononuclear variants of RS cells (sometimes termed Hodgkins cells) also were recognized to be present in most cases but were not felt to be diagnostic of HD in the absence of the more characteristic multinucleate forms. The appropriate cellular background was considered a generally heterogeneous mixture of cytologically benign inflammatory cells, which could include small lymphocytes, histiocytes, eosinophils, neutrophils, and plasma cells (see Figure 202). This era of morphologic diagnosis of HD culminated in the Rye Classification (Table 201), produced at a meeting of hematopathologists in Rye, New York, in 1966.9 The Rye Classification, with its four familiar categories of lymphocyte predominance (LP), mixed cellularity (MC), lymphocyte depletion (LD), and nodular sclerosis (NS) types, provided the standard for the diagnosis and classification of HD for over three decades. A further refinement in the definition of HD came in the 1980s, when immunohistochemistry was used to demonstrate that the RS cells typically displayed a characteristic immunophenotype. The RS cells and variants could usually be shown to express both CD30 and CD15 (typically in a membrane-and-Golgi staining pattern) (Figure 203) but usually lacked markers associated with nonHodgkins lymphomas, such as leukocyte common antigen (LCA; CD45/CD45RB), B-cell antigens (eg, CD20), and T-cell antigens (eg, CD3) (Table 202).7 CD30 and CD15 were hardly specific to HD. CD30, although originally identified on RS cells,

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Figure 201. Reed-Sternberg cell from case 2 of Thomas Hodgkins original 1832 seven-case series. (Courtesy of Dr. W. Rosenau, Dept. of Pathology, University of California, San Francisco.) (Hematoxylin and eosin; 1,000 original magnification.)

had also been shown to be expressed by activated T and B lymphocytes, some non-Hodgkins lymphomas, monocytes/macrophages, certain carcinomas, and some malignant melanomas.10,11 CD15 had been identified on activated T lymphocytes, interdigitating reticulum cells, myeloid cells, and many adenocarcinomas.10,11 But the combined expression of these two antigens on cells resembling RS cells

(and lacking T-cell and B-cell markers) had not been observed in other neoplasms. It served as a relatively specific method of diagnosis and provided the second pathologic definition of HD. This combined morphologic and immunophenotypic diagnosis of HD was reflected in the 1994 Revised EuropeanAmerican Lymphoma (REAL) Classification11 (Table 203) and is still widely used today.

Figure 202. Diagnostic Reed-Sternberg cells. A, The classic bilobed cell is no more diagnostic than B, cells having multiple nuclear lobes. Background cells are benign inflammatory cells and typically include mature small lymphocytes, histiocytes, large lymphocytes, eosinophils, and plasma cells. (Hematoxylin and eosin; 1,000 original magnification.)

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Table 201. HODGKINS DISEASE RYE CLASSIFICATION (1966) Lymphocyte predominance Mixed cellularity Lymphocyte depletion Nodular sclerosis

Although an important milestone in diagnosis, this use of immunophenotyping in diagnosis also made it clear that HD, defined morphologically, was heterogeneous and included many entities in which the candidate RS cells were CD30 and/or CD15negative. Lymphocyte predominance HD, in particular, fell out as a distinct entity, whose unusual RS cells (Figure 204) were usually CD30- and CD15 negative but positive for LCA and B-cell antigens, thus appearing more closely related to the nonHodgkins lymphomas than to the other subtypes of HD12 (Table 204). (The non-LP subtypes of HD are now frequently referred to as classic HD to distinguish them from this less reputable family member.13,14) Other cases previously diagnosed as HD also were found immunophenotypically to be better classified as non-Hodgkins lymphomas.15 This problem was particularly acute among cases diagnosed as LD HD by morphology alone, where at least one retrospective analysis revealed most of these cases to be

entities other than HDmost typically nonHodgkins lymphomas.15,16 The non-Hodgkins lymphomas most frequently misdiagnosed as HD by morphology alone were anaplastic large cell lymphoma, T-cell-rich large B-cell lymphoma, mediastinal large B-cell lymphoma, and pleomorphic peripheral T-cell lymphoma.17 For a time in the late 1980s and early 1990s, disease entities such as these were removed from the category of HD with such frequency that one could wonder whether anything would remain of HD once all the morphologic imitators had been stripped away. The differentiation of these mimics from classic HD will be considered in more detail in the section on differential diagnosis below. PATHOGENESIS The etiology of HD remains unknown. Epstein-Barr virus (EBV) has been suspected of playing a role in the pathogenesis of HD, beginning with epidemiologic studies showing an apparent link with EBV infection.18,19 Epstein-Barr virus subsequently has been found to latently infect the RS cells in the majority of cases of classic HD. Because latent infection by EBV can cause up-regulation of the bcl-2 gene, thereby allowing the cell to avoid apoptosis, the virus has long been suspected of playing a

Figure 203. Reed-Sternberg cells showing positive staining for CD30 in the characteristic membrane-and-Golgi staining pattern. (CD30 immunoperoxidase; 1,000 original magnification.)

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Table 202. REED-STERNBERG CELL PARAFFIN SECTION IMMUNOPHENOTYPE (CLASSIC HODGKINS DISEASE) CD30 CD15 CD45 CD20 T-cell antigen + +/ /+

Table 203. HODGKINS DISEASE REAL CLASSIFICATION (1994) Lymphocyte predominance Mixed cellularity Lymphocyte depletion Nodular sclerosis Lymphocyte-rich classic*

Proportion of cases showing positive staining at least focally:+ >90%; +/ 5090%; /+ 1050%; <10%.

*Provisional entity.

role in the pathogenesis of HD.20,21 The major problem with invoking EBV as etiologic in HD is the fact that the virus is absent in approximately 50 percent of cases, even when analyzed by sensitive techniques such as in polymerase chain reaction (PCR).20,21 Some have speculated that EBV could transiently infect RS cells in such virus-negative cases but then be eliminated from the cell in a mechanism termed hit-and-run.22 It is equally possible however, that an etiologic agent other than EBV is involved in such cases, or that EBV is not etiologic in any cases of HD.22,23 Latently infected B cells may simply provide better targets for the true transforming agent, causing EBV to be carried along in progeny cells as a passenger virus. A detailed discussion of the complex relationship between EBV and

HD is beyond the scope of this chapter, but the interested reader is referred to several excellent recent reviews of this subject.2224 Many lymphoid neoplasms have been found to have consistent chromosomal translocations involving cellular oncogenes, which appear to be etiologic.2530 Such characteristic karyotypic abnormalities have not been described in HD, however.31 The study of chromosomal abnormalities in HD has been hindered by the difficulty in obtaining pure populations of RS cells and propagating them in tissue culture. The few studies describing successful karyotyping of RS cells report complex hyperdiploid to near-tetraploid karyotypes with numerous structural abnormalities, but few consistent patterns.3134 A recent study using the technique

Figure 204. L&H Reed-Sternberg variants from a case of nodular lymphocyte predominance Hodgkins disease. These cells characteristically show much less nuclear atypia than the RS cells of classic Hodgkins disease. Their lobate appearance leads to their colloquial designation as popcorn cells. (Hematoxylin and eosin; 1,000 original magnification.)

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multicolor fluorescence in situ hybridization (spectral karyotyping) of RS cell lines has demonstrated chromosomal structural rearrangements clustering in ribosomal DNA regions of chromosomes 5q and 9p.35 These rearranged areas frequently appeared exported to other chromosomal sites in the form of jumping translocations.35 Such involvement of transposable DNA elements could explain the complexity and lack of uniformity seen in standard RS cell karyotypes. A final area of investigation in the pathogenesis of HD is the relationship between the RS cells and the surrounding reactive and inflammatory cells. No other malignant neoplasm features such a prominent inflammatory cell component, and there is some evidence to implicate these cells in both the development and maintenance of the neoplastic RS cell population.3641 These cell-cell interactions are highly complex and are only now becoming well understood, but appear to involve antigen presentation by RS cells resulting in an exuberant but essentially anergic host response, permitting the neoplastic cells to evade immune destruction. A complete review of this subject is beyond the scope of this chapter, but several excellent recent reviews of the subject are available.3642 Recent reports describing frequent mutations of the IB gene (which plays a central role in the normal regulation of inflammatory responses) in HD cases may represent the first recurrent molecular abnormality identified in RS cells.4345 MOLECULAR PATHOLOGY AND THE MYSTERY OF THE CELL OF ORIGIN The era of immunophenotyping that began in the 1980s, and the occasional difficulty encountered in distinguishing HD from its morphologic mimics, emphasized that there was little to define the entity of HD other than the peculiar immunophenotype of its characteristic RS cells. Even the cell of origin remained undetermined for classic HD, and there was continued speculation that it may represent a reactive condition (at least in some cases) rather than a true neoplasm.46,47 A B-cell or T-cell lymphoid origin had long been suspected, given that CD30 and CD15 could be expressed by activated lymphocytes in some cases, and B-cell and T-cell

Table 204. CLASSIC HODGKINS DISEASE VS. LYMPHOCYTE PREDOMINANCE HD PARAFFIN SECTION IMMUNOPHENOTYPE CHD CD30 CD15 CD45 CD20 T-cell Ag Epithelial membrane antigen + +/ /+ LP HD /+ + + +

Proportion of cases showing positive staining at least focally: + >90%; +/ 5090%; /+ 1050%; <10%.

antigens could be demonstrated on RS cells in rare cases, however convincing proof was lacking.47 In the 1980s and early 1990s, much attention focused on attempts to demonstrate clonal B- and T-cell lymphoid antigen receptor (ie, immunoglobulin and T-cell receptor) gene rearrangements in cases of HD.48 Such attempts, if successful, could simultaneously confirm a lymphoid origin and a clonal/neoplastic nature for the cells in HD. Studies using standard Southern blotting to detect lymphoid antigen receptor gene rearrangements generally demonstrated clonal rearrangements, usually of immunoglobulin genes, in a small percentage of cases (typically < 10%).48 But it remained unclear whether the large percentage of negative cases were true negatives or merely reflected the use of an insufficiently sensitive method (given that Southern blotting can only detect clonal cells down to a level of about 1 percent of total cells, which is approximately the proportion of RS cells in many cases of HD). This led to subsequent investigations using the more sensitive PCR technique to look for immunoglobulin gene rearrangements in tissue sections. By using PCR, several groups of investigators have demonstrated clonal immunoglobulin gene rearrangements in 32 to 50 percent of cases of classic HD, with even higher rates seen in cases where B-cell antigen expression could be detected on the RS cells.4953 The negative cases in these studies could potentially be explained as false negatives, given that immunoglobulin PCR typically employs consensus primers, which may fail to detect a significant proportion of rearrangements. However, these studies, which used DNA extracted from whole tissue sections, could not confirm that the rearrangements

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were localized to the RS cells and could not exclude the possibility that the positive cases might represent contamination (a frequent and vexing problem in PCR studies). In an attempt to definitively resolve the issue of clonal gene rearrangements in RS cells, a group of researchers at the University of Cologne led by Klaus Rajewsky undertook the Herculean task of plucking multiple individual CD30-positive RS cells from histologic sections of classic HD and subjecting the individual cells to PCR for immunoglobulin heavy chain (IgH) gene rearrangements (Figure 205). In each case where a PCR product was obtained from a cell (indicating the presence of a rearrangement), it was sequenced to determine whether it was identical to other such rearrangements found in the same case (indicating clonality of the RS cells) or whether each was different (indicating polyclonal rearrangements). In a series of cases reported in five separate publications,5458 clonal IgH rearrangements were detected in 16 of 17 cases (94%) of classic HD. Other investi-

gators using similar single-cell PCR assays have also found clonal IgH rearrangements, with the largest such series reported by a group led by Harald Stein at the Free University of Berlin59 and a smaller number of cases reported from the University of Nebraska.60 At least initially, these two latter groups reported a lower proportion of cases with clonal IgH rearrangements, as well as some cases with polyclonal IgH rearrangements.59,60 In addition, a few other groups failed to detect any rearrangements at all.61,62 It now appears that the lower rate of detection of clonal IgH by these groups was due to the use of less specific consensus PCR primers, rather than the multiple family-specific primers used by the Cologne group, and that their polyclonal re-arrangements were due to contamination by free DNA leached from polyclonal B cells in the histologic sections.6366 When Steins Berlin group repeated their analyses using family-specific primers and techniques to avoid PCR product contamination, they reported finding clonal IgH rearrangements in 24 of

Figure 205. A CD30-stained Reed-Sternberg cell removed by micromanipulation from a histologic tissue section for use in single-cell IgH gene rearrangement studies by PCR. (Reproduced with permission from Kuppers R, et al. Hodgkins disease: Hodgkin and Reed-Sternberg cells picked from histologic sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development. Proc Natl Acad Sci U S A 1994;91(23):109626.)

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25 cases, with no evidence of polyclonal re-arrangements in any case.6567 Their results appear to provide important confirmation that classic HD is a true clonal neoplasm derived from B lymphocytes in nearly all cases. In addition, there are at least three reported cases of classic HD where single-cell IgH PCR assays have demonstrated the same IgH clone present in clinical specimens from different sites in the same patient (eg, primary and relapsed tumor).57,68,69 If one accepts the standard definition of a malignancy as a neoplasm that either locally invades or distantly metastasizes, then classic HD has been proven to be a malignant tumor of B-cell origin in at least these cases, solving a century-old mystery as to the cell of origin. For these reasons, the World Health Organization (WHO) committees drafting a new lymphoma classification system have recommended a name change to Hodgkins lymphoma70 (a term appearing with increasing frequency in the literature),66,71 although the synonym of Hodgkins disease will continue to be recognized as an acceptable alternative.72 (Several excellent reviews of this fascinating subject are available for the interested reader.)7376 Beyond indicating a B-cell origin, the abovedescribed single-cell IgH PCR studies have provided evidence to suggest that the RS cells of classic HD are derived from germinal center B cells that have undergone unusual crippling mutations of their immunoglobulin genes, rendering them nonfunctionalproviding a fascinating clue as to why this B-cell neoplasm appears so different from all others.54 During normal B-cell development, nave B lymphocytes (which have few or no mutations in their immunoglobulin genes) enter germinal centers where, upon exposure to antigen, they undergo high rates of mutation of their immunoglobulin gene variable regions.76,77 This process, termed somatic hypermutation, is a key process in generating a diverse antibody repertoire and serves to increase the likelihood that high-affinity antibody is produced against a foreign antigen.77 Only B cells that are successful in creating immunoglobulin with high affinity for the antigen presented are permitted to leave the germinal center to become either plasma cells or memory B cells. B cells that cannot make antibody, or make antibody with no or low

affinity for the antigen in question, undergo apoptosis and die. Indeed, it appears likely that a default cell-death program is activated as the B cells enter the germinal center, and cells are rescued from apoptosis only if they produce high-affinity antibody; this rescue appears to involve activation of the bcl-2 anti-apoptosis gene. Sequence data from the single-cell IgH PCR assays from Rajewskys group indicate that the RS cells in their studies of classic HD have high levels of immunoglobulin gene mutations, indicating that they have been exposed to the germinal center environment.5456,77 However, in a high proportion of cases studied, the IgH genes appear to have undergone crippling mutations (eg, the creation of a stop codon in the middle of the gene), which would make it impossible for the cells to synthesize immunoglobulin.5456,77 In further support of this, studies have shown that IgH messenger ribonucleic acid (mRNA) cannot be detected in RS cells even if their IgH genes are found to be rearranged.78 This lack of IgH mRNA production due to gene mutation was confirmed by Steins Berlin group, who found in their cases that this genetic crippling could be due to either structural gene mutations or functional defects in Ig gene regulatory elements.67 How germinal center B cells incapable of immunoglobulin production escape apoptotic death is uncertain, and elucidation of this mystery may provide important insights into the pathogenesis of HD. Similar single-cell IgH PCR assays performed in cases of LP HD have led to near-universal consensus that the peculiar RS variants (termed L&H variants) in this disorder contain clonal immunoglobulin gene rearrangements,56,7981 confirming earlier suspicions based on immunophenotyping that LP HD is a B-cell neoplasm. Like classic HD, the IgH genes in LP cases show evidence of somatic hypermutation, which appears to be ongoing in the lives of these cells (ie, there is apparent acquisition of additional mutations at high rates in subsequent tumor cell generations, leading to the development of tumor subclones).80,81 Unlike classic HD however, these IgH rearrangements appear productive (ie, no crippling mutations), and, indeed, IgH mRNA and even surface immunoglobulin have been detected in a majority of cases of LP HD in some studies.78,82,83

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The apparent common origin from germinal center B cells of both classic and LP HD appears to re-establish some ties of kinship between these two disorders, which otherwise had appeared to have little in common in recent years (Figure 206). Controversy and mysteries persist in the field of HD, however. The same German investigators who demonstrated nonfunctional IgH gene rearrangements in most HD cases recently described a case of classic HD in which the RS cells harbored unmutated, potentially functional immunoglobulin heavy and light chain immunoglobulin genes, suggesting derivation from a nave, pre-germinal center B cell.84 Although these authors suggest the possibility that such cases could represent germinal center founder cells that have yet to acquire somatic mutations (thus preserving the possibility of a germinal center origin for all cases of HD), it raises questions of whether such a tumor would differ biologically from its more common crippled cousins, and how it

would differ from B-cell non-Hodgkins lymphomas. The same group also has recently reported a case of classic HD in which the RS cells, although morphologically typical and positive for both CD30 and CD15, contained clonally rearranged T-cell receptor -chain genes, with no rearrangement of immunoglobulin chain genes.84 They use this evidence to argue for a T-cell origin of HD in some cases. Other authorities, however, state that evidence of T-cell origin can be used to exclude the diagnosis of classic HD, restricting that diagnosis only to cases with RS cells consistent with germinal center B-cell origin.13 In addition, reports continue to be published of HD cases in which the RS cells appear to have more in common with dendritic cells than lymphocytes.85,86 Arguments concerning which genetically different entities should be included in the category of classic HD may be largely semantic. However, such controversies make it clear that, even today, we still lack a universally accepted definition of HD, and biologic

Figure 206. B-cell lymphomas of both Hodgkins and non-Hodgkins and the stages of normal B-cell development from which they are thought to derive. Both classic and lymphocyte predominance types of Hodgkins disease are thought to be derived from germinal center B cells. (Reproduced with permission from Kuppers R. Identifying the precursors of Hodgkin and Reed-Sternberg cells in Hodgkins disease: role of the germinal center in B-cell lymphomagenesis. J Acquir Immune Defic Syndr 1999;21 Suppl 1:749.)

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differences, if any, between these genetically different contenders to the title remain unknown. HISTOPATHOLOGY AND DIAGNOSIS The current pathologic classification schemes for HD have changed little from the widely accepted Rye Classification first proposed in 1969. The 1994 REAL Classification of Lymphoid Neoplasms (see Table 203) and the 2001 WHO Classification of Hematopoietic and Lymphoid Tumors87,88 (Table 205) both add only the entity of lymphocyte-rich classic (LRC) HD (designated a provisional entity in the REAL) to the four Rye categories of LP, MC, LD, and NS HD.9 Lymphocyte-rich classic HD was added to emphasize that a predominance of background small lymphocytes is one of the least important features in the diagnosis of LP HD. As discussed above, LP HD has many significant differences from the other classic forms of HD, and its distinction from these other subtypes is of primary importance. In the following discussion of the histopathology and differential diagnosis of the various subtypes of HD, extensive references will not be given as the features mentioned are described in detail in the standard references of these disorders,6,7,11,70,87,88 as well as recent authoritative reviews of the subject,13,42,71,89,90 which, in turn, contain references to the original literature reports. Details concerning the immunophenotype of HD and its morphologic mimics are available in these standard references as well as in a number of excellent comprehensive reviews of hematopoietic markers that have been published recently.9194 Classic Hodgkins Disease The appellation of classic Hodgkins disease includes all forms of HD containing morphologically typical diagnostic RS cells and mononuclear variants that display the characteristic CD30-positive and (usually) CD15-positive immunophenotype. It excludes only the category of LP HD. In the proposed WHO classification, there are four subtypes of classic HD: MC, LRC, LD, and NS.

Mixed Cellularity Hodgkins Disease and Lymphocyte-Rich Classic Hodgkins Disease

Mixed cellularity Hodgkins disease (MC HD) occurs most commonly in adults, and is rare in children. Any lymphoid body site may be involved, and advancedstage presentations are more common than in LP HD or NS HD, with tumor not infrequently involving the spleen, liver, and bone marrow. Purely extranodal presentations are uncommon, however. The overall prognosis is also worse than in LP or NS HD but varies with stage, and the disease can be cured in many patients. Mixed cellularity HD is the most common diagnosis made in cases of classic HD that do not show the distinctive features of the NS subtype described below. At low power, the involved tissue in both of these forms of classic HD appears diffusely effaced by a mixed cellular infiltrate consisting of scattered diagnostic RS cells and mononuclear variants in a background of benign inflammatory cells, which consists of varying proportions of mature small lymphocytes, large lymphocytes, histiocytes, eosinophils, and plasma cells (see Figure 202). Typically, small lymphocytes and eosinophils are the most numerous, but the proportions vary widely from case to case, and even common constituents such as eosinophils may be completely absent in a given case. Cases in which the background cells are virtually all small lymphocytes are termed LRC HD (Figure 207), but, as previously noted, this serves principally to draw a distinction between this form of classic HD and true LP HD. There is little evidence to suggest a biologic difference from MC HD, and most of these cases were likely diagnosed as MC HD in the past by most hematopathologists. As in the LP subtype, the histiocytes may aggregate to form granulomas, which may be quite prominent in particular cases. In lymph nodes showing early or

Table 205. HODGKINS DISEASE WHO CLASSIFICATION (2000) Nodular lymphocyte predominance HD Classic HD Mixed cellularity Lymphocyte depletion Nodular sclerosis Lymphocyte rich classic

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partial involvement by HD, tumor cells tend to accumulate in the interfollicular regions, sparing the lymphoid follicles and producing the illusion of normal nodal architecture at low power. Such cases have been termed interfollicular HD by some authors, but most cases likely represent simply early or partial nodal involvement by MC HD or other forms of classic HD.95 Except perhaps in some cases of NS HD, immunophenotyping is required to confirm the diagnosis of classic HD and exclude morphologic mimics, as will be discussed in more detail in the section on differential diagnosis below. In most cases of classic HD, the RS cells and mononuclear variants will express both CD30 and CD15, often in a characteristic membrane-and-Golgi staining pattern, although in some individual cases either the membrane pattern or Golgi pattern may predominate (see Table 202). The majority of classic HD cases also lack evident expression of LCA (CD45) and T-cell or B-cell markers. CD30 expression is seen in virtually all cases and is taken by many to be a required element for the diagnosis of any form of classic HD. CD15, on the other hand, is recognized to be absent in roughly 20 percent of classic HD cases. Approxi-

mately 20 percent of classic HD cases will also show expression of B-cell markers, such as CD20, by the RS cells, but in contrast to B-cell non-Hodgkins lymphomas, the CD20 expression in classic HD tends to be relatively weak and focal. Expression of T-cell antigens such as CD3, however, appears to be distinctly unusual in classic HD. Some authorities hold that such expression can be used as grounds to exclude the diagnosis,13 whereas others believe that Hodgkins disease may truly be of T-cell origin in rare cases.96 A variety of other antigens are expressed by RS cells, but few offer any advantages in specificity or sensitivity over those described above.9194
Lymphocyte Depletion HD

Lymphocyte depletion Hodgkin disease (LD HD) is the least commonly diagnosed form of HD, at least in industrialized nations. This may reflect a lack of clear criteria to separate it from other forms of classic HD, as well as a high standard imposed by many pathologists for the diagnosis, given the history of misdiagnosis of other entities as LD HD in the pre-immunophenotyping era (see below). It is more common in patients infected with HIV than in the general popu-

Figure 207. Hodgkins disease, lymphocyte-rich classic type. The phenotype and distribution of the Reed-Sternberg cells are similar to that in mixed cellularity Hodgkins disease, but virtually all the background cells are small lymphocytes. The phenotype of the RS cells and their marked nuclear atypia distinguish this entity from lymphocyte predominance Hodgkins disease. (Hematoxylin and eosin; 1,000 original magnification.)

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lation and more common in industrialized societies than in developing countries. Patients with LD HD are also more likely to be elderly and present with involvement of abdominal lymph nodes, liver, spleen, and bone marrow. They often lack peripheral lymphadenopathy. Overall, the prognosis is poor but stage for stage appears similar to that of MC HD. Morphologically, LD HD resembles the MC subtype, but has a significantly higher proportion of RS cells and mononuclear variants relative to inflammatory cells. The RS cells and variants may be present in sheets resembling a diffuse large cell nonHodgkins lymphoma or other poorly differentiated large-cell neoplasm (Figure 208). The tumor cells may also be elongated, resembling at least focally the spindled cells of a malignant sarcoma (Figure 209). Given this resemblance to other large-cell malignancies, it is perhaps not surprising that retrospective review of cases diagnosed as LD HD by morphology alone in the pre-immunophenotyping era have revealed that the majority of such cases actually represented entities other than classic HD (most commonly diffuse large B-cell lymphomas).15,16 The distinction between MC HD with numerous RS cells and LD HD is sometimes an arbitrary one.

There are no established criteria concerning the precise proportion of RS cells at which MC HD becomes LD HD. This fact, combined with the skepticism with which the diagnosis of LD HD became viewed at the onset of the immunohistochemical era, has led some hematopathologists to diagnose most cases of classic HD not showing the characteristic features of the NS subtype as MC HD.
Nodular Sclerosis Hodgkins Disease

Of all the subtypes of classic HD, NS is the most distinctive. Patients tend to be younger, with most patients presenting as adolescents or young adults. However, the disease can occur at any age. In contrast with both the LP and MC subtypes, the disease occurs predominantly in females, with the mediastinum being the single most common site of involvement. Prognosis depends on the stage of disease at presentation, but many patients can be cured. Like other forms of classic HD, diagnostic RS cells and mononuclear variants are present and often relatively numerous. However, the hallmark of NS HD is the presence of dense bands of sclerosis, which tend to divide the cellular infiltrate into nod-

Figure 208. Hodgkins disease, lymphocyte depletion type. Reed-Sternberg cells and mononuclear variants outnumber background inflammatory cells, and can mimic large cell non-Hodgkins lymphomas as well as other poorly differentiated large-cell malignancies. (Hematoxylin and eosin; 1,000 original magnification.)

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ules of varying size (Figure 2010). When these sclerotic bands and nodules are well developed, the diagnosis of NS HD can be suspected even at low power examination. When the sclerotic bands and nodular architecture are poorly developed, caution should be exercised to prevent mistaking a residual lymph node trabecular sinus or band of fibrinous exudate as sclerosis. Polarized light examination will reveal refringent collagen in areas of true sclerosis. In NS HD, however, other distinctive histologic features are also generally present to suggest the diagnosis. These include a relatively high proportion of neutrophilic inflammation (which may include microabscesses) and scattered apoptotic (or mummified) RS cells. One will also typically observe, in formalin-fixed tissues, apparent retraction of the cytoplasm of RS cells and variants to produce socalled lacunar variants of RS cells (Figure 2011). Although none of these features is specific for NS HD, their presence should spur the search for bands of sclerosis. Some pathologists term cases of classic HD showing these latter features, but lacking obvious sclerosis, as the cellular phase of NS HD, particularly when it occurs in a clinical setting characteristic of NS HD. However, there is little evidence to indicate a natural progression of NS HD from nonsclerotic to sclerotic phases in most cases.

Another distinctive morphologic feature that occurs in some cases of NS HD is the presence of RS cells and variants in diffuse sheets (at least focally), producing the so-called syncytial variant (Figure 2013). In addition to causing confusion with large cell non-Hodgkins lymphomas and other large-cell malignancies, the presence of RS cells in sheets appears to be associated with a worse prognosis.13 The syncytial variant of NS HD appears to overlap significantly with the high-grade or grade 2 category of NS HD as defined by the British National Lymphoma Investigation, which has also been associated with poor clinical outcome.97 Lymphocyte Predominance Hodgkins Disease The MC, LD, NS, and LRC subtypes are collectively termed classic HD, in recognition of the presence in all four of highly pleomorphic diagnostic RS cells that typically display the characteristic CD30-positive and CD15-positive immunophenotype. In contrast, LP HD contains few, if any, diagnostic RS cells. Instead, scattered among the background small lymphocytes is a peculiar form of giant cell termed L&H RS variants (a name taken from the 1966 Lukes-Butler classification,98 in which this entity was

Figure 209. Hodgkins disease, lymphocyte depletion type. Reed-Sternberg cells and mononuclear variants are present in vaguely spindled shapes, mimicking a malignant sarcoma. (Hematoxylin and eosin; 400 original magnification.)

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Figure 2010. Hodgkins disease, nodular sclerosis type. Bands of dense sclerotic collagen separate the cellular infiltrate into nodules of varying size. (Hematoxylin and eosin; 40 original magnification.)

Figure 2011. Hodgkins disease, nodular sclerosis type. In formalin-fixed tissue sections, the Reed-Sternberg cells and mononuclear variants frequently demonstrate artifactual retraction of their cytoplasm from surrounding cells to produce lacunar variants. (Hematoxylin and eosin; 1,000 original magnification.)

Figure 2012. Hodgkins disease, nodular sclerosis type, syncytial variant. Reed-Sternberg cells and mononuclear variants are present in large aggregates and sheets, mimicking diffuse large cell non-Hodgkins lymphoma. (Hematoxylin and eosin; 400 original magnification.)

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termed the lymphocytic and/or histiocytic subtype of HD). Morphologically, these giant cells resemble lobate histiocytes more than typical RS cells, and their multiple nuclear lobes display relatively finely dispersed chromatin and small, inconspicuous nucleoli (see Figure 204). This characteristic nuclear lobation results in their being colloquially termed popcorn cells. Their bland nuclear cytology and scant cytoplasm make them relatively inconspicuous, and they may not be readily apparent at screening magnification, but a diligent search at high power will reveal their presence. The great majority of cases of LP HD will have a nodular architecture at low power, with aggregation of the background small lymphocytes into densely packed nodules resembling those of follicular lymphoma (see Figure 2013). These cases are often termed nodular LP HD (NLP HD). Lack of any nodular architecture in a large biopsy should call the diagnosis of LP HD into question.99 Histiocytes are also frequently present in the cellular background of LP HD and not uncommonly form scattered granulomas, which can be a clue to the diagnosis. Lymphocyte predominance HD can present at any age, including childhood, although most cases occur in adults. Generally, peripheral lymph nodes are affected, and involvement of the mediastinum is uncommon. Involvement of extranodal sites is distinctly uncommon. Disease is usually localized at

presentation, and the prognosis is excellent in lowstage cases, although late relapses may occur. Progression of LP HD to large B-cell lymphoma may also occur in some cases. Immunophenotyping is not necessary to confirm the diagnosis of LP HD if the above-described characteristic morphologic features are present. If immunohistochemical stains are performed, a number of differences from classic HD will be observed. The peculiar L&H variants of LP HD will be positive for CD30 only in a minority of cases, and generally not in the membrane-and-Golgi staining pattern seen in cases of classic HD. CD15 expression is seen only rarely and should raise some doubt as to the accuracy of the diagnosis. Repeated studies have documented expression of both LCA and B-cell markers such as CD20 by the L&H RS variants, but these stains can be difficult to interpret in routine practice, given that the majority of the surrounding small lymphocytes are B cells that will also express these antigens. The B-cell nature of the background small lymphocytes is another feature, however, that distinguishes the LP subtype from classic HD, where the majority of the non-neoplastic lymphoid cells are T cells. The few small T lymphocytes present in LP HD are described by some authors as frequently forming rings or rosettes around the L&H cells; CD57 expression by these T cells has been

Figure 2013. Hodgkins disease, lymphocyte predominance type. At low power, a nodular architecture will be found at least focally in virtually all cases. (Hematoxylin and eosin; 400 original magnification.)

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advocated by some as a diagnostic aid, but others have reported it to be of limited utility. L&H RS variants are also reported to stain for epithelial membrane antigen (EMA) in the majority of cases, another feature distinguishing these cells from the RS cells of classic HD. DIFFERENTIAL DIAGNOSIS Many entities can mimic HD pathologically. Tumor giant cells histologically indistinguishable from the RS cells of classic HD can be observed in tumors as varied as diffuse large cell non-Hodgkins lymphomas, malignant melanoma, large cell carcinomas, and pleomorphic sarcomas. Thus, morphologic RS cells, although necessary for the diagnosis of classic HD, are hardly sufficient. The problem is compounded by the presence of rare cases of bona fide classic HD in which the RS cells and variants may appear relatively bland cytologically, raising the question of whether they represent benign reactive large lymphocytes (centroblasts or immunoblasts) rather than neoplastic lymphoid cells. The distinctive subtype of LP HD also raises its own particular differential diagnosis. Although a differential diagnosis can be generated for each of the five subtypes of HD, it is simpler and more practical to consider the entities that most commonly mimic HD in its three basic morphologic

patterns: classic HD with scattered single RS cells, classic HD with aggregates of RS cells, and LP HD. Classic Hodgkins Disease with Scattered Single Reed-Sternberg Cells Most cases of HD appear histologically as scattered single diagnostic RS cells or mononuclear variants in a background of benign-appearing inflammatory cells. Reed-Sternberg cells in these cases may vary in density from area to area and form rare small clusters but are not present in large aggregates or sheets. Forms of HD with this appearance would include MC HD, LRC HD, and the nonsyncytial forms of NS HD. Many entities other than HD are capable of producing this pattern, some of which may mimic classic HD to perfection morphologically. Each of these entities should be carefully considered in the differential diagnosis before a diagnosis of classic HD is rendered. Several non-Hodgkins lymphomas may, on occasion, be histologically indistinguishable from MC HD or other forms of classic HD with scattered RS cells (Figure 2014). Principal among these is anaplastic large cell lymphoma (ALCL). Anaplastic large cell lymphoma has had a long and colorful history. Originally described as Ki-1-positive large cell lymphoma,100 it was characterized by pleomorphic

Figure 2014. Anaplastic large cell lymphoma. Atypical giant cells resembling the Reed-Sternberg cells of classic Hodgkins disease are present. (Hematoxylin and eosin; 1,000 original magnification.)

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CD30-positive tumor giant cells resembling RS cells and variants that lacked CD15 and frequently expressed T-cell antigens. The resemblance to RS cells extended to the CD30 staining pattern, which frequently marked both cell membrane and Golgi region. These tumors were often found to display other features not typically observed in HD, such as cohesive growth of tumor cells in lymph node sinuses, although such sheet-like growth is not seen in all cases. Later, it was discovered that a subset of ALCL cases, which were virtually always of T-cell origin genetically, contained a distinctive t(2;5) translocation that caused expression of a NPM-ALK fusion protein that could, in turn, be recognized by the ALK1 antibody and predicted a favorable clinical outcome in systemic cases.101103 With these findings focusing attention on this distinctive T-cell form of ALCL, and few data to suggest biologic differences between B-cell large cell lymphomas that expressed CD30 versus those that did not, the term ALCL became largely restricted to tumors of T-cell type.13,101 With the discovery that most cases of classic HD were of probable germinal center B-cell origin (see above), a further wedge was driven between these two entities. Today most authorities suggest that classic HD can be distinguished from ALCL in most cases by application of immunoperoxidase stains that exploit the above-described differences13,71 (Table 206). Positive staining of the candidate RS cells for CD15 or CD20 (if weak and focal) favors classic HD. Positive staining of the large cells for the ALK protein, T-cell antigens, or LCA (CD45) favors ALCL. Epithelial membrane antigen expression is also found more commonly in ALCL than in HD, whereas expression of fascin (which to

Table 206. CLASSIC HODGKINS DISEASE VS. ANAPLASTIC LARGE CELL LYMPHOMA PARAFFIN SECTION IMMUNOPHENOTYPE CHD CD15 CD20 CD45 T-cell antigen Epithelial membrane antigen ALK/t(2;5) + /+ ALCL +/ +/ + +/

Proportion of cases showing positive staining at least focally: + >90%; +/ 5090%; /+ 1050%; <10%.

date cannot be tested in paraffin sections) appears relatively specific to HD. Morphologic clues with ALCL include the presence of so-called hallmark cells (with eccentrically placed horseshoe or kidneyshaped nuclei) (Figure 2015), the presence of tumor giant cells with wreath-like nuclei (Figure 2016),104 nucleoli that appear less prominent than in HD, and tumor cells that grow in cohesive sheets, often with a sinusoidal pattern apparent at least focally. Clinical clues with ALCL would include tumor presenting in a child or young adolescent and an extranodal presentation. Even with extensive immunophenotyping, however, rare cases cannot be clearly classified as either ALCL or HD; these will be discussed in the section on gray zone lymphomas below. If the pattern of dense sclerosis and nodule formation is well developed, and the RS cells and variants express the characteristic CD30/15-positive immunophenotype, few other entities need be considered in the differential diagnosis of NS HD. In the early 1990s, there were reports of cases of ALCL that could mimic the characteristic morphology and immunophenotype of NS HD105; these were included in the category of ALCL, Hodgkins-like that appeared as a provisional entity in the 1994 REAL classification.11 This category has been dropped from the proposed WHO classification, however,87 and there appears to be a consensus among experts that most cases of ALCL and CHD can be distinguished using the guidelines outlined above.13,25 Diffuse large B-cell lymphoma (DLBCL) may also mimic MC HD and other forms of classic HD with scattered RS cells, particularly when it contains numerous admixed inflammatory cells in the variant known as T-cell/histiocyte-rich large B-cell lymphoma. In these cases, the large B cells cited above as potential mimics of the L&H cells of LP HD can also resemble classic RS cells and thus mimic MC HD. Lack of CD30 and CD15 staining, combined with the strong uniform CD20 expression that would be expected of large B cells in such cases, will help to exclude classic HD.106 Peripheral T-cell lymphoma (PTCL) can contain pleomorphic large cells present in a background of small lymphocytes and thus mimic MC HD. Morphologically, one may note a so-called continuum of atypia in such PTCL cases, that is, a spectrum of

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Figure 2015. Anaplastic large cell lymphoma. Sheet-like growth and the presence of tumor cells with reniform to horseshoe-shaped nuclei (hallmark cells) provide clues that this is not classic Hodgkins disease. (Hematoxylin and eosin; 1,000 original magnification.)

atypical small, atypical intermediate, and atypical large lymphoid cells, rather than the sharp distinction between the atypical large RS cells/variants and the benign-appearing background inflammatory cells characteristically observed in classic HD. Immunophenotyping will generally reveal the T-cell nature of

the neoplastic cell population in such cases. Several facts about the immunophenotype of PTCLs that may trip up the unwary should be kept in mind, however. First, loss of one or more pan-T-cell antigens by PTCLs is not uncommon, and thus the tumor may appear to have a null cell phenotype if only one

Figure 2016. Anaplastic large cell lymphoma. A large atypical tumor cell is present with a wreath-like nucleus, which is characteristic of anaplastic large cell lymphoma. (Hematoxylin and eosin; 1,000 original magnification.)

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T-cell antigen is assayed. Second, CD15 is expressed by up to 60 percent of peripheral T-cell lymphomas. Finally, CD30 can be expressed by a variety of T-cell lymphomas in addition to classic ALCL. Whether a T-cell lymphoma co-expressing CD30 and CD15 should be considered a T-cell variant of classic HD is largely an issue of semantics. But such a tumor would clearly differ from the more common germinal-center B-cell type of CHD, a point that should be made clear in the pathology report in such rare cases. Most authorities at present would regard the expression of one or more T-cell antigens as an argument against the case being considered classic HD. Mixed cellularity HD and similar forms of classic HD can also be mimicked by an unusual type of B-cell proliferation known as post-transplant lymphoproliferative disorder (PTLD). Post-transplant lymphoproliferative disorders are EBV-induced B-cell proliferations that may arise following either solid organ or bone marrow transplantation.107 They are an unusual class of disorders that span the spectrum from benign to malignant, and their behavior is difficult to predict from their pathologic appearance.107 Polymorphic PTLDs are destructive lesions that efface the architecture of involved tissues, which may be lymph nodes or extranodal sites. Morphologically, they most closely resemble a diffuse mixed small and large cell lymphoma such as a marginal zone lymphoma. Some cases, however, may include scattered atypical large cells resembling RS cells, which may express CD30. Staining for CD20 or other B-cell markers will reveal strong uniform positive staining of both large and small cells, however, essentially excluding HD. Lymph nodes responding to antigens that provoke T-cell immunity, such as viral infections and vaccinations, may undergo expansion of the interfollicular areas, which on high-power microscopic examination may contain numerous reactive large lymphocytes (centroblasts and/or immunoblasts), a condition known as interfollicular immunoblastic hyperplasia. In its milder forms, the bland cytology and general preservation of nodal architecture raise little concern of malignancy, but more florid cases may include atypical large lymphoid cells and thus mimic either early interfollicular HD or MC HD. Contributing to this potential confusion is the notinfrequent expression of CD30 by reactive immuno-

blasts, particularly in response to viral infections.108 The cytologic atypia in such cases generally falls short of that typically observed in classic HD, and the expression of CD30 and CD15 in a membraneand-Golgi pattern is seldom observed. Consultation with the clinician in such equivocal cases may yield important clinical information, for example, that the patients lymphadenopathy is resolving spontaneously or that the patient has serologic evidence of infectious mononucleosis. Finally, the possibility of LP HD must be considered in cases of MC HD with relatively bland RS cells and a predominance of small lymphocytes and histiocytes in the inflammatory background. This particular differential diagnosis between LP HD and classic HD will be discussed in more detail below. Classic Hodgkins Disease with Aggregates of Reed-Sternberg Cells In two forms of classic HD, LD HD and the syncytial variant of NS HD, RS cells and variants may be present in large aggregates or diffuse sheets. This may cause the diagnosis of classic HD to not be considered in the initial differential diagnosis. The presence of such subtypes and variants of CHD raises differential diagnostic considerations not applicable to more characteristic forms of classic HD such as MC HD. The differential diagnosis of LD HD is somewhat broader than that of MC HD, but many of the same entities must be considered. The closer resemblance of LD HD to other poorly differentiated large-cell malignancies such as non-small cell carcinoma, malignant melanoma, large cell non-Hodgkins lymphoma, and pleomorphic sarcomas means that such entities must generally be excluded by immunohistochemical stains for epithelial, melanocytic, T/Bcell, and other mesenchymal antigens. Absence of staining for such lineage-associated markers, combined with characteristic membrane-and-Golgi staining for CD30 and CD15, confirms the diagnosis. Morphologically, LD HD does not typically demonstrate the dense cohesion of tumor cells typical of many other poorly differentiated large-cell malignancies, but this is an unreliable feature. In CD15-negative cases, anaplastic large cell lymphoma would need to be excluded, following the guidelines estab-

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Figure 2017. T-cell-rich large B-cell lymphoma. This variant of diffuse large B-cell lymphoma contains scattered large B cells in a background of numerous mature small T cells, and may thus mimic classic Hodgkins disease. Immunophentotyping is required to confirm the diagnosis. (Hematoxylin and eosin; 1,000 original magnification.)

lished in the discussion of MC HD above. The differential diagnosis would also have to include the syncytial variant of NS HD, which will also have RS cells present in diffuse sheets. The presence of sclerotic bands of collagen excludes LD HD and con-

firms NS HD, which typically presents in younger patients. Cases lacking sclerosis but showing other characteristic features of NS HD could be considered the cellular phase of NS HD, particularly if present in a young patient (see below).

Figure 2018. Peripheral T-cell lymphoma. Such lymphomas may contain pleomorphic large cells resembling the Reed-Sternberg cells of classic Hodgkins disease and may show some immunophenotypic features of that disorder. The continuum of atypical cells, from small to intermediate to large, helps distinguish this entity from Hodgkins disease. (Hematoxylin and eosin; 1,000 original magnification.)

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In cases of presumed NS HD where the sclerotic banding pattern is poorly developed or the immunophenotype is not classic for HD (eg, lack of CD15 expression), essentially all the entities described in discussion of the differential diagnosis of MC HD should be considered as outlined above. In the case of a presumed syncytial variant of NS HD, the differential should be broadened further to include the other entities discussed in the differential diagnosis of LD HD, and the possibility of processes such as metastatic melanoma or carcinoma might need to be excluded. A particular problem is distinguishing NS HD from the subtype of diffuse large B-cell lymphoma known as primary mediastinal (thymic) large B-cell lymphoma. Both commonly present as mediastinal masses in young women, so the clinical setting can be identical. Moreover, both tumors feature large, atypical, lymphoid cells separated by fibrous bands. However, the pattern of fibrosis is much finer in mediastinal large B-cell lymphoma than in NS HD, surrounding small clusters of tumor cells in a pattern termed compartmentalizing fibrosis, as opposed to the coarse fibrous bands and large tumor nodules seen in NS HD (Figure 2019). To further complicate matters, primary mediastinal large B-cell lymphoma is frequently CD30 positive.109 However, the strong uniform staining of the B-cell lymphoma for CD20 generally serves to distinguish the two. Lymphocyte Predominance Hodgkins Disease Lymphocyte predominance HD can be confidently diagnosed on the basis of its morphologic appearance alone if all of the characteristic diagnostic features discussed above are present. Certain other conditions, both benign and malignant, may mimic LP HD in some instances, however, and care must be taken to exclude them from the differential diagnosis. Cases of LP HD in which the L&H cells display higher degrees of nuclear atypia may be confused with classic Hodgkins disease, but the morphologic and immunophenotypic features described above usually suffice to exclude classic HD, which rarely displays the regular, densely packed, small cell-predominant, nodular architecture seen in most

cases of LP HD. The typical case of LP HD is much more likely to be confused with small cell B-cell non-Hodgkins lymphomas, particularly follicular lymphoma. The low-power microscopic appearance of LP HD in its most common nodular form can mimic a low-grade follicular B-cell lymphoma to perfection (Figure 2020), and both entities should be considered in the pathologic differential before either is diagnosed. B-cell immunostains will also demonstrate the majority of the cells in these nodular aggregates to be mature small B cells in both cases, adding to the confusion. Distinction between these entities is made principally by high-power microscopic examination, which will reveal the majority of the small lymphocytes present to have round nuclear contours in LP HD, whereas a significant population of centrocytes (small cleaved cells) will be evident in cases of true follicular lymphomas (Figure 2021). Further high-power examination will reveal the presence of the characteristic lobate L&H cells in cases of LP HD. Lymphocyte predominance HD must also be distinguished from a form of florid reactive follicular hyperplasia of lymph nodes known as progressive transformation of germinal centers (PTGC). Progressive transformation of germinal centers may be found in the lymph nodes of patients with LP HD either preceding, concurrent with, or subsequent to the diagnosis of HD, and in some cases LP HD appears to arise from a background of PTGC. However, a connection between these two entities is far from clear, and PTGC is too commonly found as a focal feature of nonspecific reactive follicular hyperplasia to be a precursor lesion to LP HD in all cases. Progressive transformation of germinal centers is characterized by the presence, in a background of typical follicular lymphoid hyperplasia, of scattered, strikingly enlarged lymphoid follicles four to five times the size of surrounding reactive follicles, which appear to consist mainly of small cells and lack a typical germinal center. Such transformed follicles resemble the individual nodules of NLP HD, but in a typical case of PTGC the transformed follicles are widely scattered, not tightly clustered with effacement of nodal architecture as is typically observed in NLP HD. In cases of PTGC where transformed follicles appear focally clustered and the diagnosis of incipient NLP HD is considered, a diligent search for L&H cells is warranted.

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Figure 2019. Mediastinal diffuse large Bcell lymphoma. This variant of diffuse large Bcell lymphoma may mimic classic Hodgkins disease its clinical presentation, its sheet-like growth (mimicking the syncytial variant of NS HD), and in its frequent expression of CD30. The pattern of fine compartmentalizing fibrosis and the strong diffuse expression of CD20 help to confirm the diagnosis. (Hematoxylin and eosin; 1,000 original magnification.)

Figure 2020. Low-grade follicular nonHodgkins lymphoma. The low-power appearance of multiple, crowded, ill-defined follicles mimics the nodular appearance of nodular lymphocyte predominance Hodgkins disease. (Hematoxylin and eosin; 40 original magnification.)

Figure 2021. Low-grade follicular nonHodgkins lymphoma. The presence of numerous small cleaved lymphocytes (centrocytes) and the absence of L&H variants excludes the diagnosis of LP HD. (Hematoxylin and eosin; 1,000 original magnification.)

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Lymphocyte predominance HD may also be confused with diffuse small B-cell lymphomas, such as small lymphocytic lymphoma, mantle cell lymphoma, and marginal zone lymphoma (ie, MALToma or monocytoid B-cell lymphoma), particularly if the characteristic nodular architecture of LP HD is not well developed. Small cell lymphoma will almost invariably demonstrate its characteristic pseudofollicular architecture due to the presence of proliferation centers containing larger prolymphocytes and paraimmunoblasts, which are absent from cases of LP HD. Mantle cell and marginal zone lymphomas can be distinguished by the absence of the characteristic L&H cells at high power, but caution is urged, particularly in the case of marginal zone lymphomas, which may contain large numbers of histiocytes and large lymphocytes resembling L&H cells to a certain extent. One can decrease the likelihood of confusing mantle cell and marginal zone lymphomas with LP HD if one is extremely cautious in the diagnosis of LP HD in cases entirely lacking its characteristic nodular architecture. If biopsy material is too limited to assess architecture with confidence, a definitive diagnosis is often best avoided. Finally, the scattered large L&H cells present among the small lymphocytes and histiocytes of LP HD may mimic the morphologic variant of diffuse large B-cell lymphoma known as T-cell/histiocyterich large B-cell lymphoma. As the name implies, this tumor features large numbers of admixed reactive small T lymphocytes or histiocytes (Figure 2017). Several reports describe a relatively aggressive clinical course in these patients. The large B cells in these cases may resemble L&H variants of LP HD; however, the lack of nodular architecture and a paucity of background small B cells would argue against the diagnosis of LP HD. A case of LP HD which is entirely diffuse should call into question the diagnosis of LP HD (ie, should make one suspect the diagnosis is incorrect).99 Grey Zone Lymphomas In the vast majority of cases, the question of whether a tumor containing candidate RS cells represents HD can be solved by routine paraffin section immunoperoxidase staining, where a CD30-positive, CD15-

positive, and T/B-cell antigen-negative immunophenotype will be found in most cases of true classic HD. Most cases of classic HD will also demonstrate a combination of characteristic clinical presentation, typical morphology, and characteristic immunophenotype. As a general rule of thumb, the diagnosis of classic HD can be made with relative confidence if at least two of these three features are present. For example, a case of HD presenting in an extranodal site (an uncommon clinical presentation) is still classic HD, provided that it demonstrates diagnostic RS cells in an appropriate mixed inflammatory background and these cells show the characteristic CD30/15-positive phenotype in a membrane-and-Golgi staining pattern. Any lack of the characteristic morphology or immunophenotype in such a case, however, should cause the diagnosis of HD to be made only with extreme caution. Similarly, an adolescent female with a mediastinal mass showing microscopic dense sclerosis and lymphoid nodules containing scattered large lymphoid cells that are CD20 negative and CD30/15 positive in the characteristic pattern can be confidently diagnosed with NS HD, even if the large lymphoid cells are relatively bland morphologically. But if the cells instead appear CD30 negative, it should put the diagnosis of classic HD in doubt. Ancillary markers can be employed in equivocal cases. For example, as discussed above, a case on the borderline between classic HD and ALCL can be subjected to stains for ALK protein expression and fascin, in the hope of tipping the scales toward one or the other diagnosis. In addition, genetic studies can be employed. Although PCR-based gene rearrangement studies may be difficult to interpret (given that clonal B-cell populations could be detected in both classic HD and B-cell lymphomas), the presence of a strong clonal band in standard Southern blots for T- or B-cell antigen receptor genes can be used as evidence to support the diagnosis of a non-Hodgkins lymphoma as these are not generally found in classic HD. In rare cases, however, even using all the strategies outlined above, it may be impossible to exclude pathologic mimics and reach an unequivocal diagnosis of HD. This may be due to lack of material for needed studies (eg, lack of snap-frozen tissue for fascin stains or Southern blotting) or to completely equivocal results. A classic example of the latter

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would be a tumor with RS-like cells and mononuclear variants, present both as single scattered cells and in clusters, in which the neoplastic large cells stain only with CD30 in a membrane-and-Golgi pattern. Such a tumor could reasonably be considered either a CD15-negative case of classic HD or an ALK-negative case of ALCL. A Southern blot could fail to yield a clonal gene rearrangement in either case: between 1 and 5 percent of lesional cells must carry the rearrangement to be detected by Southern blotting, and both classic HD and ALCL may have too few neoplastic cells in some cases. Tumors such as those described above have been termed gray zone lymphomas110 and, although rare, present a situation frustrating to both pathologists and clinicians. There is some evidence to suggest that such lymphomas may respond equally well to therapy for HD and non-Hodgkins lymphomas, leaving clinicians free to treat such neoplasms according to their best clinical judgment.111 The wider availability of molecular biologic techniques to look for the presence of the non-productively rearranged, crippled immunoglobulin chain genes typically identified by research laboratories in most cases of classic HD analyzed to date may help resolve many such gray zone cases, but likely not all. The ability to distinguish between HD and its many morphologic mimics in all cases will require a universally accepted genetic definition of HD and technology that is cost-effective and widely available to diagnostic pathologists. REFERENCES
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