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The biology of Hodgkins lymphoma

Ralf Kppers

Abstract | Hodgkins lymphoma was first described in 1832. The aetiology of this lymphoma, however, remained enigmatic for a long time. Only within the past 10 years has the B-cell nature of the pathognomonic Hodgkin and ReedSternberg (HRS) cells been revealed, along with several recurrent genetic lesions. The pathogenetic role for EpsteinBarr virus infection has also been substantiated. HRS cells in classical Hodgkins lymphoma have several characteristics that are unusual for lymphoid tumour cells, and the Hodgkins lymphoma microenvironment is dominated by an extensive mixed, potentially inflammatory cellular infiltrate. Understanding the contribution of all of these changes to the pathogenesis of this disease is essential for the development of novel therapies.
More than 150 years ago Thomas Hodgkin described several cases of a disease which was subsequently named Hodgkins disease1. The cells that are a hallmark of this disease the mononucleated Hodgkin cells and the multinucleated ReedSternberg cells were first described by Dorothy Reed and Carl Sternberg around 19002,3. As it was realized that this disease is a lymphoid malignancy, it was renamed to Hodgkins lymphoma4. On the basis of differences in the morphology and phenotype of the lymphoma cells and the composition of the cellular infiltrate, Hodgkins lymphoma is subdivided into classical Hodgkins lymphoma and nodular lymphocyte-predominant Hodgkins lymphoma (NLPHL). NLPHL accounts for about 5% of cases. Classical Hodgkins lymphoma is further subdivided into nodular sclerosis, mixed cellularity, lymphocyte depletion and lymphocyte-rich Hodgkins lymphoma. For example, nodular sclerosis Hodgkins lymphoma, which accounts for ~60% of cases of Hodgkins lymphoma, is characterized by extensive fibrotic bands separating nodules containing Hodgkin and Reed Sternberg (HRS) cells, whereas such bands are missing in mixed cellularity Hodgkins lymphoma, which accounts for about 30% of cases and is characterized by a prominent mixed cellular infiltration. Hodgkins lymphoma is one of the most frequent lymphomas in the Western world, with an incidence of about 3 cases per 100,000 people per year. Treatment of Hodgkins lymphoma is a success story: current treatment protocols involve multi-agent chemotherapy and/or radiotherapy and achieve cure rates of 8090% (Ref. 5). The pathognomonic HRS cells in classical Hodgkins lymphoma and the HRS cell variants in NLPHL, which are known as lymphocytic and histiocytic (L&H) cells, usually account for only about 1% of cells in the tumour tissue, although some cases may show more than 10% HRS cells (fIG. 1). The majority of cells in Hodgkins lymphoma lesions are a mixed infiltrate of various types of cells of the immune system. The rarity of HRS and L&H cells is one of the reasons for the difficulty in their characterization. Moreover, few cell lines have been established from Hodgkins lymphoma patients. However, important progress has been made in recent years to reveal the nature and pathogenesis of HRS and L&H cells. The following discussion will focus on these exciting findings.

The cellular origin of L&H and HRS cells Both L&H and HRS cells are derived from B cells they share characteristics with normal, mature B cells that have been exposed to antigen.
Maturation of normal B cells. When mature B cells are activated by binding of antigen to their B-cell receptor (BCR) and co-stimulation by antigen-specific T helper (TH) cells, they migrate into B-cell follicles of secondary lymphoid organs such as lymph nodes, spleen and Peyers patches and start to vigorously proliferate. This leads to the development of histological structures called germinal centres6. Besides proliferating B cells, the germinal centres harbour TH cells, follicular dendritic cells and other types of dendritic cells, and macrophages. The proliferation of the germinal centre B cells is accompanied by the process of somatic hypermutation, which generates point mutations and some deletions and duplications at a high rate, specifically in the variable (V) region genes of immunoglobulin (Ig) heavy and light chains7. Through this mechanism, variants of the BCR are generated.
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Institute of Cell Biology (Tumour Research), University of Duisburg-Essen, Medical School, Virchowstrasse 173, 45122 Essen, Germany. e-mail: ralf.kueppers@ uk-essen.de doi:10.1038/nrc2542 Published online 11 December 2008

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At a glance
Hodgkin and ReedSternberg (HRS) cells of classical Hodgkins lymphoma are probably derived from germinal centre B cells that have acquired disadvantageous immunoglobulin variable chain gene mutations and normally would have undergone apoptosis, whereas lymphocytic and histiocytic (L&H) cells of NLPHL appear to derive from antigen-selected germinal centre B cells. Few cases of classical Hodgkins lymphoma originate from T cells. Classical Hodgkins lymphoma is unique among human lymphomas in the extent to which the lymphoma cells have undergone reprogramming of gene expression. They have lost expression of most B cell-typical genes and acquired expression of multiple genes that are typical for other types of cells of the immune system. Multiple signalling pathways and transcription factors show deregulated activity in HRS cells, including nuclear factor-B, JakStat, PI3KAkt, Erk, AP1, notch 1 and receptor tyrosine kinases. The transforming events involved in the generation of HRS cells are only partly understood, but several known recurrent genetic lesions involve members of the nuclear factor-B or JakStat signalling pathways. HRS cells attract many cells into the lymphoma tissue, resulting in a typical inflammatory microenvironment. This environment probably promotes the survival of HRS cells and helps them to escape attack from cytotoxic T or natural killer cells.

indicated that these cells resemble an intermediate developmental stage between germinal centre and memory B cells15. HRS cell origins. The origin of HRS cells in classical Hodgkins lymphoma was more difficult to interpret, as HRS cells show an immunophenotype that is unlike any other cell of the haematopoietic system, with coexpression of markers of several lineages. However, these cells carry rearranged and somatically mutated Ig V genes in nearly all cases13,16. Interestingly, in about onequarter of cases of classical Hodgkins lymphoma, clearly destructive somatic mutations were found that rendered originally functional Ig V region genes non-functional (for example, nonsense mutations)13,16,17. Such mutations happen in germinal centre B cells, but normally cause the immediate death of these cells. This indicates that HRS cells in these cases derive from crippled, pre-apoptotic germinal centre B cells16,18. It is possible that HRS cells in general originate from germinal centre B cells that have acquired unfavourable mutations and were prone to undergo apoptosis, as only a fraction of disadvantageous mutations can be identified by sequence analysis. For example, replacement mutations causing reduced affinity of the BCR, cannot be easily identified by sequence analysis, but are probably a major cause of apoptosis induction in germinal centre B cells. However, because Ig gene rearrangements have also been found in T-cell and myeloid malignancies, it has been questioned whether the detection of Ig V gene rearrangements indeed proves the B-cell nature of HRS cells. However, additional evidence unequivocally shows the B-cell nature of these cells. First, complete Ig heavy and light chain rearrangements are only found in B cells13. Second, somatic mutations occur only in antigen-activated B cells, and the somatic mutations in the rearranged V genes of HRS cells are, at least initially, selected for functionality, which can only happen if the BCR is expressed by the cell16,19. Third, Hodgkins lymphoma cell lines have undergone Ig class switching, a further process that only occurs in antigen-activated B cells, and there are indications that primary HRS cells have also undergone class-switch recombination20,21. Fourth, composite lymphomas consisting of a Hodgkins lymphoma and a mature B-cell lymphoma are frequently clonally related and carry somatically mutated Ig V genes with both shared and distinct mutations, demonstrating that both lymphomas derive from a common precursor: a mature germinal centre B cell22,23. Analysis of cases of classical Hodgkins lymphoma with expression of T-cell markers revealed that a fraction of these carried T-cell receptor gene rearrangements and lacked Ig gene rearrangements2428. Thus, it appears that rare cases of Hodgkins lymphoma derive from T cells. There is, however, the caveat that some T-cell lymphomas may mimic Hodgkins lymphoma, with co-expression of CD30 (also known as tumour necrosis factor receptor superfamily member 8 (TNFRSF8)) and CD15 by the lymphoma cells, which is normally taken as a strong argument for a diagnosis of classical Hodgkins lymphoma29. Notably, a gene expression study of cell lines from a T-cell-derived
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Many mutations will be disadvantageous for the cells, for example, if they cause premature stop codons, loss of the correct reading frame, gain of autoreactivity or a reduction of the affinity of the antibody for the cognate antigen. Normally, such cells efficiently undergo apoptosis and are taken up by macrophages6. Germinal centre B cells that acquire affinity-increasing mutations will be positively selected by interaction with follicular dendritic cells and antigen-specific follicular TH cells. Proliferation and mutation of germinal centre B cells mainly takes place in the dark zone of the germinal centre, whereas the interaction with follicular dendritic cells and TH cells mainly takes place in the light zone, where the B cells show little proliferative activity. Germinal centre B cells repeatedly migrate between the dark and light zones and undergo multiple rounds of proliferation, mutation and selection, before they finally differentiate into either memory B cells or plasma cells8. Many germinal centre B cells also perform class-switch recombination, by which the isotype of the Ig heavy chain is changed so that antibodies with different effector functions are generated. The origin of L&H cells. L&H cells in NLPHL express multiple B-lymphocyte markers, including germinal centre B-cell differentiation antigens such as the transcription factor BCL-6, and activation-induced cytidine deaminase, the key enzyme for somatic hypermutation and class switching 9,10. L&H cells also grow in a follicular pattern that resembles the structure of a germinal centre. These features indicate that L&H cells are derived from germinal centre B cells11. This was validated by the analysis of isolated L&H cells for Ig V gene rearrangements. These studies revealed that L&H cells are clonal populations of tumour cells with rearranged, functional and somatically mutated Ig V genes, with a fraction of cases showing ongoing somatic hypermutation1214. Thus, L&H cells probably derive from antigen-selected germinal centre B cells. A gene expression study of isolated L&H cells

Plasma cell
A terminally differentiated B cell that is specialized for the secretion of antibodies.

Class switching
The somatic recombination process by which immunoglobulin isotypes are switched from IgM to IgG, IgA or Ige.

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a b c

Figure 1 | Hodgkin and reedSternberg (HrS) cells in their microenvironment. Nature Reviews | Cancer This figure illustrates the typical histological and immunohistochemical picture seen in classical Hodgkins lymphoma. a | Haematoxylin and eosin staining of a case of nodular sclerosis Hodgkins lymphoma. Several large HRS cells are visible, one of them in the centre of the picture. b | CD30 immunostaining (red) highlighting several large CD30+ HRS cells. c | CD3 staining showing the dominant T-cell infiltrate and a ring of T cells around an HRS cell in the centre.

Hodgkins lymphoma (HDLM2), from B-cell Hodgkins lymphoma and from other B- or T-cell lymphomas, including CD30+ anaplastic large T-cell lymphoma, revealed that the T-cell Hodgkins lymphoma line clustered together with the other Hodgkins lymphoma lines30. This supports the existence of T-cell Hodgkins lymphoma cases and suggests that HRS cells originating from B or T cells acquire a similar gene expression programme following malignant transformation.

Deregulated transcription factor networks B-cell lymphomas and leukaemias usually retain key phenotypical and functional features of the B cells they derive from31. HRS cells are the most striking exception to this rule, as they show a global loss of their B-cell phenotype32. A partial downregulation of the B-cell phenotype is also seen in L&H cells15. With few exceptions, HRS cells retain only B-cell features associated with antigen-presenting functions and interaction with TH cells, such as expression of major histocompatibility class II, CD40 (also known as TNFRSF5) and CD80 (Refs 33,34). This suggests that communication with CD4+ TH cells is important for HRS cells, as will be discussed later. In addition to the downregulation of B cell-specific molecules, HRS cells often express markers of other haematopoietic cell lineages, such as T-cell markers (CD3 and CD4), cytotoxic molecules (granzyme B), dendritic cell markers (fascin and CC chemokine 17 (CCL17)) or myeloid markers (colony-stimulating factor 1 receptor and 1-antitrypsin)3538. We are now beginning to understand the factors that are involved in this reprogramming. Multiple transcription factors that activate the expression of numerous B-cell genes are not expressed in HRS cells, including OCT2 (also known as POu2F2), BOB1 (POu2AF1) and Pu.1, which explains or at least contributes to the lack of expression of their target genes3941 (fIG. 2). Many B-cell genes are further inactivated by epigenetic mechanisms, such as DNA methylation42,43. Moreover, early B-cell factor 1 (eBF1), which is one of the key factors for the B-cell differentiation programme, is expressed only at a low level in HRS cells35,44. In the mouse, eBF1 not only activates the transcription of B cell-specific genes, but also suppresses the expression of myeloid and T-cell genes45. Thus,
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eBF1 acts as a B-lineage commitment factor, and its low level of expression in HRS cells may contribute to the deregulated expression of myeloid and T-cell genes. TCF3 (also known as E2A), which encodes the two helixloophelix transcription factors e12 and e47, is detectable in HRS cells, but its function is impaired by two of its inhibitors: activated B-cell factor 1 (ABF1, also known as musculin) and inhibitor of differentiation and DNA binding 2 (ID2)35,46,47 (fIG. 2). The physiological role for ABF1 in haematopoietic cells is poorly understood. Notably, however, ID2 is normally expressed in dendritic cells and natural killer cells and supports development of these cells, but it suppresses B-cell generation48,49. PAX5, the main B-cell lineage commitment and maintenance factor, is expressed in HRS cells, although many of its direct target genes are downregulated50. PAX5 promotes the expression of many B-cell genes and suppresses genes of other haematopoietic lineages51. The functional impairment of PAX5 is probably not due to mutations in the gene32. As many B-cell genes are regulated by the coordinated action of several transcription factors, the absence or impaired function of other B-cell transcription factors may partly explain the downregulation of PAX5 target genes in HRS cells. The T-cell transcription factor notch 1 is another negative regulator of the B-cell programme that is highly expressed in HRS cells52. Notch 1 is probably activated in HRS cells by stimulation through its ligand jagged 1, which is produced by cells in the Hodgkins lymphoma microenvironment53. Moreover, HRS cells have downregulated deltex 1, an inhibitor of notch 1 (Ref. 53). Notch 1 plays a key part in lymphoid development by promoting T-cell differentiation and inhibiting B-cell development. Notch 1 reduces the expression of e12 and e47 and eBF1 and induces the transcription of ABF1 (Ref. 53). Moreover, notch 1 binds to PAX5 in Hodgkins lymphoma cell lines, which might impair PAX5 function53. STAT5A and STAT5B (signal transducer and activator of transcription 5A and 5B) were identified as further transcription factors that are potentially involved in the reprogramming of HRS cells 54. STAT5A and STA5B are active in HRS cells, and expression of a constitutively active form of STAT5A or STAT5B in normal B cells induces a phenotype resembling that of HRS cells, including upregulation of CD30 and GATA3 and downregulation of BCR expression54. The downregulation or functional impairment of transcription factors that suppress the expression of non-Blineage genes and the aberrant expression of transcription factors that suppress B-cell genes and induce the expression of genes typical for other haematopoietic cell types explains not only the global loss of the B-cell phenotype, but also why HRS cells show a heterogeneous expression of many genes of T cells, dendritic cells and others. Deregulated expression of key transcription factors of haematopoietic stem cells (HSCs) may further contribute to the reprogramming of HRS cells. GATA2, which is expressed by HRS cells in about half of Hodgkins lymphoma cases but not by normal mature B cells, is required for the proliferation and survival of HSCs, and also has a role in mast cell development 55. Notably, GATA2
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LMP2A LMP1

STAT5A and STAT5B ? HRS cell

BMI1

B-cell gene

ID2 E12 or E47

ABF1 E12 or E47 M M M

B-cell gene

It is an intriguing question whether the reprogramming of the HRS cells is functionally associated with their origin from pre-apoptotic germinal centre B cells. It is possible that HRS precursor cells that acquire a non-B-cell gene expression programme might thereby evade the selectional forces that act on germinal centre B cells and hence be selected for on the basis of survival. It remains to be clarified whether this reprogramming is associated with the specific transforming events involved in the generation of HRS cells.

B-cell gene

Deltex 1 EBF Notch 1-IC ? Notch 1 E12 or E47 PAX5 OCT2 PU.1

BOB1

Figure 2 | reprogramming of Hodgkin and reedSternberg (HrS) cells. HRS cells are unique in the extent to which they have lost their B-cell phenotype. We are starting to understand the mechanisms that have a role in this reprogramming. Deregulated expression of inhibitors of B-cell molecules (inhibitor of differentiation and DNA binding Nature Reviews | Cancer 2 (ID2), activated B-cell factor 1 (ABF1) and notch 1), downregulation of B-cell transcription factors (OCT2, BOB1 and PU.1) and epigenetic silencing of B-cell genes (CD19 and immunoglobulin H (IgH)) seem to be involved in this process. ID2 binds to the B-cell transcription factors E12 and E47, encoded by E2A, and inhibits their binding to DNA. Heterodimers of ABF1 and E12 or E47 can still bind to DNA, but are unable to activate B-cell genes. Notch 1 inhibits both E12 and E47, and the early B-cell factor (EBF), which is expressed at a low level in HRS cells, and perhaps also PAX5. Notch 1 activity is mediated through ligands expressed in the Hodgkins lymphoma microenvironment and by downregulation of the notch 1 inhibitor deltex 1. Further factors that are implicated in the downregulation of B-cell genes are the EpsteinBarr virus-encoded latent membrane proteins LMP1 and LMP2A, the polycomb group gene BMI1 and active STAT5A and STAT5B. STAT5A and STAT5B are presumably induced by signalling through the interleukin 21 (IL-21) receptor. IC, intracellular.

expression is induced by notch 1 (Ref. 56). An important group of HSC regulators are polycomb group (PcG) proteins, which repress transcription through chromatin modification and regulate the self-renewal of HSCs. HRS cells express members of the two main PcG protein complexes: BMI1, RING1, polyhomeotic-like protein (also known as HPH), chromobox protein 8 (also known as HPC3) and MeL18 (complex 1), and eeD and eZH2 (complex 2)5759. BMI1 and eZH2 are also expressed by subsets of normal germinal centre B cells, although normally in a mutually exclusive pattern58. RyBP (RING1 and yy1-binding protein), another PcG member, is also frequently expressed by HRS cells, but is not detectable in normal B cells59. PcG proteins can downregulate B-cell genes, and HSC and common lymphoid precursors show promiscuous co-expression of genes of multiple haematopoietic lineages6062. Thus, the acquisition of HSC or common lymphoid precursor features by HRS cells may also contribute to their expression of markers of different haematopoietic lineages.
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Genetic lesions and deregulated signalling The rarity of HRS and L&H cells hampered not only the clarification of their cellular origin but also the identification of genetic lesions involved in their generation. Several pathogenetic mechanisms have been revealed using molecular cytogenetic techniques and molecular analysis of microdissected HRS and L&H cells. As rescue of HRS cells from apoptosis is probably a key event in Hodgkins lymphoma pathogenesis, activators or inhibitors of apoptosis were studied for genetic aberrations in HRS cells. Mutations in TP53, FAS, caspase 8, caspase 10, FAS-associated via death domain (FADD), BCL-2 agonist of cell death (BAD) or ataxia telangiectasia mutated (ATM), or translocations of BCL2 were found only rarely if at all in HRS cells and Hodgkins lymphoma cell lines (TABLe 1). However, analyses for TP53 mutations in primary HRS cells were restricted to mutations in selected exons, and deletions in TP53 were recently identified in Hodgkins lymphoma cell lines, so TP53 alterations in primary HRS cells may be more frequent than was anticipated from earlier studies6365. Besides the activation of numerous anti-apoptotic pathways (discussed below), important anti-apoptotic mechanisms in HRS cells involve upregulated expression of CASP8 and FADD-like apoptosis regulator (CFLAR), which inhibits FAS signalling, and XIAP (also known as BIRC4), which suppresses caspase activation6668. Translocations involving BCL-6, a main regulator of the germinal centre B-cell differentiation programme, are frequently found in L&H cells, but only rarely in HRS cells of classical Hodgkins lymphoma21,69. Interestingly, the genetic lesions most frequently found so far in HRS cells involve members of two signalling pathways: janus kinase (jak)Stat and nuclear factor-B (NF-B). Many cytokines signal through members of the jak family, which phosphorylate STAT factors on activation. The phosphorylated STATs dimerize and translocate to the nucleus where they function as transcription factors. There are frequent genomic gains of JAK2 in HRS cells, and suppressor of cytokine signalling 1 (SOCS1), a negative regulator of jakStat signalling, is often somatically mutated and inactivated in classical Hodgkins lymphoma and NLPHL7072. NF-B activity is affected by several types of genetic alterations (fIG. 3). REL, a member of the NF-B transcription factor family, shows genomic gains and amplifications in nearly half of Hodgkins lymphoma cases, and these alterations correlate with ReL protein levels7375. In rarer instances, the inhibitor of NF-B (IB) family member BCL-3, which can positively regulate NF-B activity, may be affected by genomic gains or
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Table 1 | Pathogenetic mechanisms in HRS and L&H cells
Gene HRS cells
REL NFKBIA NFKBIE TNFAIP3 BCL3 JAK2 SOCS1 TP53 MDM2 CD95 Gains, amplification Point mutations, deletions Point mutations, deletions Point mutations, deletions Gains, translocations Gains, amplifications Point mutations, deletions Point mutations, deletions Gains Point mutations Point mutations, deletions Translocations NF-B NF-B NF-B NF-B NF-B JakStat JakStat p53 p53 FAS-mediated apoptosis JakStat BCL-6 50 20 15 40 (60% EBV) 10 40 45 10 60 10 50 35

Type of alteration

Pathway

Frequency of cases with alterations (%)

L&H cells
SOCS1 BCL6

No somatic mutations were found in the following genes: NRAS, BCL10, CASP8, CASP10, FADD (FAS-associated via death domain), and BAD (BCL2-associated agonist of cell death). HodgkinReedSternberg (HRS) cells also lack chromosomal NPM (nucleophosmin)ALK (anaplastic lymphoma kinase) and BCL2immunoglobulin H translocations (with the exception of composite follicular and Hodgkins lymphomas with shared BCL2immunoglobulin H translocations). No point mutations in JAK2 (Janus kinase 2) were found. EBV, EpsteinBarr virus; L&H, lymphocytic and histiocytic; NFKBI, nuclear factor-B inhibitor; SOCS1, suppressor of cytokine signalling 1; TNFAIP, tumour necrosis factor- induced protein.

translocations76,77. NFKBIA, which encodes IB, which inhibits NF-B signalling by binding to NF-B factors in the cytoplasm and preventing their nuclear translocation, is somatically mutated in ~20% of classical Hodgkins lymphoma cases7880, and mutations affecting NFKBIE have also been found81. Recently, we identified inactivating mutations in TNF-induced protein 3 (TNFAIP3) in HRS cells of classical Hodgkins lymphoma (R.K., unpublished observations). TNFAIP3 encodes A20, which functions as a ubiquitinase and deubiquitinase of members of the NF-B signalling pathway and a negative regulator of NF-B activity. Notably, all cases with mutations that clearly prevented TNFAIP3 function were epsteinBarr virus (eBV)-negative, indicating that eBV infection and TNFAIP3 mutations are alternative mechanisms in HRS cell pathogenesis. The analysis of Hodgkins lymphoma cell lines implies that mutations in several members of the NF-B pathway may coexist in HRS cells (for example, NFKBIA and TNFAIP3 mutations in the Hodgkins lymphoma cell line KMH2), suggesting that multiple lesions cooperate in the deregulation of this pathway. This is unusual, as tumours typically show lesions in single members of a particular signalling pathway, and supports the essential role for strong and constitutive NF-B activity for HRS cells. The constitutive activity of the jakStat pathway and the NF-B transcription factors is not only caused by genetic lesions, but also by autocrine and/or paracrine signalling events. Four STAT factors STAT3, STAT5A, STAT5B and STAT6 are active in HRS cells54,8284. STAT6 appears to be activated in an autocrine fashion through expression of interleukin 13 (IL-13) and IL-13R by HRS cells85. expression and activation of STAT5A and STAT5B is
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augmented by NF-B activity in HRS cells86. Moreover, it was recently reported that co-expression of IL-21 and IL-21R by HRS cells leads to activation of STAT5A, STAT5B and STAT3 (Refs 54,87). There is, however, a controversy surrounding which of these STAT factors is the main target of IL-21 signalling54,87. Furthermore, STAT factors may be activated by receptor tyrosine kinases, many of which are expressed and activated in HRS cells (see below). Signalling events that contribute to the activation of NF-B through both the canonical and non-canonical pathways presumably involve members of the Tnfr family (fIG. 3). HRS cells express the Tnfr family members CD30, CD40, TACI (also known as TNFRSF13B), BCMA (TNFRSF17) and RANK (TNFRSF11A)33,8890. T cells expressing the CD40 ligand are often in close contact with HRS cells91. CD30 ligand is expressed by eosinophils and mast cells in the lymphoma microenvironment 92,93. However, conflicting data have been published regarding the role for CD30 in activation of NF-B and the need for ligand-binding for activity of the CD30 receptor 9296. APRIL (TNFSF13), one of the ligands for TACI and BCMA, is produced by neutrophils in the Hodgkins lymphoma microenvironment, and BAFF (TNFSF13B), the second ligand for these receptors, is expressed by HRS cells and other cells in the microenvironment 88,97. RANK may be activated in an autocrine fashion, as expression of the RANK ligand has been reported for Hodgkins lymphoma cell lines89. A further factor that probably contributes to NF-B activation in HRS cells is notch 1 (Ref. 98). A strong NF-B signature was recently also observed for L&H cells, but the mechanisms activating NF-B in these cells are not understood15. L&H cells lack expression of
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CD30L EBV infection (40%) CD40L APRIL BAFF CD40 BCMA TACI

RANKL

CD40L

CD40 RANK

CD30

LMP1

TNFAIP3 Ub mutations Ub (60% EBV ) TNFAIP3

Ub

RIP

TRAF

NIK

NEMO Classical NF-B pathway IKK IKK NFKBIA and NFKBIE mutations IB and (1020%) IB p50 p65 Proteasomal degradation

IKK IKK Alternative NF-B pathway p100 RELB

REL amplification (30%)

p50

p65

BCL3 gains or translocations (rare)

BCL-3 p50 p50 p52 RELB

Nuclear membrane

Figure 3 | nuclear factor-B (nF-B) in Hodgkin and reedSternberg (HrS) cells. HRS cells show constitutive activity of both the classical (canonical) and alternative (or non-canonical) NF-B signalling pathways, which is probably a major pathogenetic mechanism in Hodgkins lymphoma. In the classical pathway, activation of diverse receptors Nature Reviews | Cancer stimulates TRAFs (tumour necrosis factor (TNF) receptor-associated factors), which associate with the receptor interacting protein (RIP), thus activating the inhibitor of NF-B (IB) kinase (IKK) complex (composed of IKK, IKK and NEMO), which then phosphorylates IB and IB. This marks these proteins for ubiquitylation and subsequent proteasomal degradation, thereby releasing the NF-B heterodimers (p50p65 and p50REL (not shown)) and allowing their nuclear translocation. The signal transduction from the cell surface receptors to the IKK complex can be inhibited by TNF-induced protein 3 (TNFAIP3), which removes activating ubiquitins from RIP and TRAFs and adds ubiquitins to these molecules to mark them for proteasomal degradation. In the alternative NF-B pathway, receptor activation leads to stimulation of the kinase NIK (MAPK3K14), which then activates an IKK complex (and which can also activate the classical pathway). Activated IKK processes p100 precursors into mature p52 molecules, which then translocate as active p52RELB heterodimers into the nucleus. The NF-B activity in HRS cells is probably mediated by diverse mechanisms: receptor signalling through CD40, RANK, BCMA, and TACI, genomic REL amplification, destructive mutations in IKBA and IKBE and signalling through the EpsteinBarr virus (EBV)-encoded latent membrane protein 1 (LMP1). CD30 ligand (CD30L)CD30 signalling is also shown as an activating mechanism, although this is debated9296. Recently, frequent inactivating mutations in the NF-B inhibitor TNFAIP3 were identified, mainly in EBV cases (R.K., unpublished observations). HRS cells presumably also harbour nuclear BCL-3(p50)2 complexes, and in a small fraction of cases the strong BCL-3 expression may be mediated by genomic gains or chromosomal translocations. The frequency of genetic lesions and viral infections in classical Hodgkins lymphoma cases is indicated.

CD30 and, although they express CD40, it has been reported that the CD4+ T cells in direct contact with the L&H cells lack expression of CD40 ligand91,99. Besides the jakStat and NF-B pathways and deregulated transcription factors involved in the reprogramming of the HRS cells (discussed above), multiple additional signalling pathways and transcription factors show deregulated and constitutive activity in HRS cells, including PI3KAkt, the erk pathway, AP1 and multiple receptor
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tyrosine kinases100105. The PI3K pathway may be activated in HRS cells through CD30, CD40, RANK and receptor tyrosine kinases. Activity of this pathway is implicated by the detection of the active form of one of its main targets the serine/threonine kinase AKT1 and by phosphorylation of AKT1 substrates in HRS cells100,101. Inhibition of AKT1 causes death of Hodgkins lymphoma cell lines, supporting an essential role for this pathway in HRS cell survival100,101. The erk pathway, which regulates apoptosis,
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proliferation and differentiation of cells, may also be activated through CD30, CD40 and RANK in HRS cells105. Active forms of several members of the Mapk family, which are serine/threonine kinases, are expressed by HRS cells, including eRK1, eRK2 and eRK5 (Refs 103,105). Inhibition of erk activity has anti-proliferative effects for Hodgkins lymphoma cell lines105. AP1 transcription factors are dimers composed of members of the jun and Fos families. HRS cells express juN and juNB at high levels, and their nuclear localization indicates that they might be active102. Indeed, AP1 induces many target genes in HRS cells, including CD30 and galectin 1, and promotes HRS cell proliferation106,107. NF-B activity contributes to upregulation of juNB, whereas the mechanisms for upregulation of juN are not yet clear 102. Receptor tyrosine kinases are involved in the regulation of cell proliferation, survival, growth and differentiation. In numerous tumours, activating mutations of specific receptor tyrosine kinases have been described. HRS cells show aberrant expression of multiple receptor tyrosine kinases, including platelet-derived growth factor receptor- (PDGFRA), epithelial discoidin domaincontaining receptor 2 (DDR2), macrophage-stimulating protein receptor (MSPR), TRKA and TRKB104. Mutations in the genes encoding these kinases have not been found, but they are probably activated in HRS cells by autocrine and/or paracrine mechanisms104. Receptor tyrosine kinase expression is most pronounced in nodular sclerosis Hodgkins lymphoma, but is also detected at various levels in the other subtypes. expression of multiple receptor tyrosine kinases is rarely seen in other lymphomas, except primary mediastinal B-cell lymphoma108. Notably, the pathogenetic similarities between Hodgkins lymphoma and primary mediastinal B-cell lymphomas involve not only the deregulated expression of receptor tyrosine kinases but also genetic lesions in the NF-B and jakStat signalling pathways, which are also active in primary mediastinal B-cell lymphomas: gains and amplifications of REL and JAK2 and inactivating mutations in SOCS1 are also seen in this form of nonHodgkins lymphoma (reviewed in Ref. 109). There are also histological similarities between these lymphomas and, as Hodgkins lymphoma also frequently involves the mediastinum, differential diagnosis is sometimes difficult. In summary, genetic lesions identified so far in HRS cells frequently involve members of the NF-B and jak Stat signalling pathways, pointing to a central role for these pathways in Hodgkins lymphoma pathogenesis. These pathways are further activated by several external signals in an autocrine or paracrine fashion. Overall, multiple signalling pathways are active in HRS cells, and these frequently interact and cooperate. Hodgkins lymphoma in Central and South America, the association can be up to 90%, and Hodgkins lymphoma in patients with AIDS show eBV-infected HRS cells in nearly all cases112,113. In eBV+ HRS cells, three viral proteins (eBNA1 (eBV nuclear antigen 1), LMP1and LMP2A) and two non-coding RNAs (BARTs and eBeRs) are expressed. eBNA1 is expressed in all proliferating eBV-infected cells, as it is important for the replication of the viral episome and its distribution to the daughter cells. eBNA1 may also directly contribute to the pathophysiology of Hodgkins lymphoma, as it can mediate upregulation of CCL20 to attract regulatory T cells (TReg) and downregulates the protein-tyrosine phosphatase-, which is a putative tumour suppressor gene114,115. LMP1 is an oncogene, and LMP1 transgenic mice develop B-cell lymphomas116. This viral protein aggregates in the cell membrane and mimics an active CD40 receptor, thereby inducing constitutive NF-B activation117. Additionally, LMP1 can activate AP1, p38, PI3K and jakStat signalling 118. LMP2A can replace the function of a BCR, as it bears a cytoplasmic ITAM (immunoreceptor tyrosinebased activation motif) that is reminiscent of the signalling domains of the BCR119. B-cell precursors expressing LMP2A can survive without a BCR, which supports a role for LMP2A as a BCR surrogate120. Importantly, as signalling though the BCR and CD40 are two major physiological survival and selection signals for germinal centre B cells121, it appears that eBV has hijacked these pathways to mediate the survival of eBV-infected germinal centre B cells. Notably, it has also been reported that LMP1 and LMP2A contribute to the downregulation of the B-cell phenotype in eBV+ HRS cells122,123 (fIG. 2). The features of the eBV-encoded genes expressed in HRS cells point to a pathogenetic role for the virus. However, this idea is still questioned by some investigators owing to the restricted pattern of expression of eBVencoded genes (the proliferation-inducing gene EBNA2 is not expressed), the similar NF-B activity in eBV+ and eBV cases, the absence of LMP2A signalling molecules in HRS cells and the fact that 60% of Hodgkins lymphomas develop without eBV. There are several recent observations that support a key role for eBV in the virus-harbouring cases. First, there is an inverse relationship between expression of multiple receptor tyrosine kinases and eBV in HRS cells, indicating that eBV can replace pathogenetic functions that are otherwise mediated by deregulated activity of receptor tyrosine kinases124. Second, in vitro experiments revealed that eBV can indeed rescue crippled germinal centre B cells from apoptosis125127. LMP2A has a major role in this regard, as its expression is essential for the survival of growth-transformed germinal centre B cells128. Third, practically all cases of Hodgkins lymphoma with Ig V gene mutations that prevent any BCR expression are eBV+, supporting an essential role for eBV in the development of HRS cells from germinal centre B cells with these types of unfavourable mutations129. Fourth, as discussed above, there is a striking inverse relationship between mutations in TNFAIP3 and the presence of eBV in HRS cells, suggesting that eBV can provide the pathogenetic function of TNFAIP3 mutations.
VOLuMe 9 | jANuARy 2009 | 21 2009 Macmillan Publishers Limited. All rights reserved

LMP1
(Latent membrane protein 1). An eBV-encoded protein that activates Nf-B and other factors.

LMP2A
(Latent membrane protein 2A). An eBV-encoded protein that mimics a B-cell receptor.

The pathogenetic role for EpsteinBarr virus eBV is found in HRS cells in about 40% of classical Hodgkins lymphoma in the Western world, mostly in cases of mixed cellularity and lymphocyte-depleted Hodgkins lymphoma, and less frequently in nodular sclerosis and lymphocyte-rich classical Hodgkins lymphoma. eBV is rarely found in NLPHL 110,111. In paediatric

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HRS precursor cells There is currently much interest in the existence and characteristics of cancer stem cells130. In Hodgkins lymphoma (and other B-cell lymphomas), the presence of clonally rearranged and somatically mutated Ig V genes unequivocally demonstrates a derivation of the lymphoma cells from mature germinal centre or postgerminal centre B cells, and excludes a derivation from HSCs. This is further supported by the clonal relationship of composite lymphomas that was discussed earlier. However, the germinal centre or post-germinal centre B-cell origin of HRS cells does not exclude the possibility that the first transforming events in the multistep transformation process were acquired in a lymphoma precursor at an earlier differentiation stage. Nevertheless, it is possibile that a subset of the HRS cells has a higher proliferative and renewal potential than the bulk of the tumour clone, thus fulfilling the definition of cancer stem cells, and that there is a phenotypical precursor population that feeds the morphologically identifiable HRS cell population. Studies with Hodgkins lymphoma-derived cell lines showed that the mononuclear Hodgkin cells give rise to the multinucleated ReedSternberg cells by endomitosis, and that the ReedSternberg cells have little capacity for further clonal growth131,132. Hence, the pool of HRS cells is probably sustained by mononuclear Hodgkin cells, some of which undergo endomitosis and give rise to multinuclear ReedSternberg cells. The question of a phenotypical precursor population that feeds the HRS cell clone was addressed by analysing Hodgkins lymphoma tissues for the existence of CD30 non-HRS cells with genomic alterations identical to those of the respective HRS cell clones. In two cases, CD30 cells with numerical chromosomal abnormalities that were similar to those of the HRS clones were found, arguing for an HRS cell progenitor population133. However, numerical chromosomal abnormalities are not the best clonal marker, and Hodgkins lymphoma patients indeed harbour an increased frequency of bystander cells with chromosomal abnormalities134,135 that could be the origin of the abnormal CD30 non-HRS cells. Moreover, when non-HRS cells from other cases were studied for the translocations or point mutations that were detected in the HRS cells of the respective cases, no such mutations were found, although the number of cells analysed does not exclude the rare presence of such cells 70,81. Spieker et al. reasoned that in eBV + Hodgkins lymphoma, which shows a clonal infection of HRS cells by eBV, the putative phenotypical precursor cells that sustain the pool of HRS cells must also be eBV+ (Ref. 136). In a molecular analysis of eBV+ CD30 B cells from eBV+ Hodgkins lymphoma, it was found that few, if any, of the small CD30 eBV-infected B cells belonged to the HRS cell clone, arguing against the existence of a HRS precursor population in these cases136. However, it remains possible that a subset of the mononuclear Hodgkin cells has features of cancer stem cells. Given the rarity of HRS cells and the difficulties of growing them in culture or immunodeficient mice, the search for such cells will be a difficult task. The Hodgkins lymphoma microenvironment Classical Hodgkins lymphoma is characterized by an infiltration of many different types of cells of the immune system into the lymphoma tissue, including T cells, B cells, plasma cells, neutrophils, eosinophils and mast cells, such that the HRS cells themselves usually represent only about 1% of cells in the tumour. There is evidence that many of the cells are actively attracted by HRS cells (fIG. 4). For example, HRS cells secrete CCL5 (RANTeS), CCL17 (TARC) and CCL22, which attract TH2 cells and TReg cells137139. The secretion of IL-5, CCL5, CCL28 and granulocytemacrophage colony-stimulating factor by HRS cells presumably causes the recruitment of eosinophils into the Hodgkins lymphoma microenvironment137. CCL5 additionally attracts mast cells. HRS cells also secrete IL-8, which attracts neutrophils137. Notably, chemokines may not only be involved in the attraction of other cells into the lymphoma microenvironment but also have direct effects on HRS cell survival and proliferation, as was recently shown for CCL5 (Ref. 138). There is evidence that the recruitment of inflammatory cells into the lymphoma microenvironment is essential for HRS cell survival. Several observations indicate that HRS cells are dependent on survival signals received from other cells: it is difficult to grow HRS cells in culture; HRS cells do not survive in immunodeficient mice; HRS cells are rarely found in peripheral blood; and, even when they metastasize into non-lymphoid organs, they are embedded in their typical microenvironment 140. CD4+ T cells, the largest population of infiltrating non-tumour cells, are presumably particularly important, and it is noteworthy that improved therapy of patients with AIDS causing restoration of CD4+ T-cell counts resulted in an increased risk of developing Hodgkins lymphoma, whereas the incidence of other lymphomas dropped141. Some of the survival signals that are provided by inflammatory cells to the HRS cells were already mentioned: triggering of CD40 signalling by CD40L-expressing rosetting T cells, activation of TACI and BCMA through production of their ligand APRIL by neutrophils, and perhaps activation of CD30 through CD30L-expressing mast cells and eosinophils. Moreover, HRS cells express IL-3R, which has growth- and survival-promoting effects following activation, and there is evidence that HRS cells can induce activated T cells to secrete IL-3 (Refs 142,143). Some cellular interactions in Hodgkins lymphoma tissue may involve multiple players. For example, HRS cells stimulate fibroblasts through various factors (for example, TNF, transforming growth factor- (TGF) and fibroblast growth factors)144,145, and the activated fibroblasts in turn produce eotaxin and CCL5, thus contributing to the attraction of eosinophils and TReg cells into the lymphoma145. HRS cells also orchestrate their cellular microenvironment to evade an attack by cytotoxic T cells or natural killer cells (fIG. 4). A considerable fraction of infiltrating CD4+ T cells are TReg cells, which have been shown to have immunosuppressive activity on Hodgkins lymphoma-infiltrating cytotoxic T cells146,147. The presence of a large population of TReg cells in the Hodgkins
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Endomitosis
Chromosomal replication without cell division.

Rosetting T cells
A phenomenon in which CD4+ T cells surround HRs cells in a rosette pattern.

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Eosinophil

Attraction through secretion of eotaxin and CCL5 Attraction through secretion of IL-5, CCL5 and CCL28 Fibroblast Stimulation by secretion of TNF_ and TGF` Inhibition through PD1L and secretion of IL-10, TGF`, galectin 1 CD30L Attraction through secretion of CCL5 CD30 CD30 CD30L

Mast cell

CD4+ TH cell CD54 CD40 MHC II CD80 BCMA TRKA CD18 or CD11A CD40L TCR CD28 Attraction through secretion of TARC, CCL5 and CCL22 APRIL Granulocyte Expansion stimulated by galectin 1 TRKA stimulation through NGF secretion

HRS cell
PD1L

PD1 Attraction through secretion of TARC, CCL5, CCL20 and CCL22

CD8+ TC cell Inhibition through secretion of IL-10

CD4+ TReg cell

TH1 response

A T-helper-1-cell-mediated immune response is mediated by pro-inflammatory cytokines such as IfN, IL-1 and TNf. It promotes cellular immune responses against intracellular infections and malignancy.

Figure 4 | Cellular interactions in the Hodgkins lymphoma microenvironment. The figure shows interactions between Nature Reviews | Cancer Hodgkin and ReedSternberg (HRS) cells and other cells in their microenvironment, both direct cellular interactions and those with soluble mediators. Multiple cytokines and chemokines are involved in the attraction of the various types of cells into the Hodgkins lymphoma microenvironment. CD4+ T helper (TH)-cells are attracted by HRS cells through secretion of the chemokines TARC, CC chemokine 5 (CCL5) and CCL22. These, as well as CCL20, also attract TReg cells. CCL5 has an additional role in the recruitment of eosinophils and mast cells. Eosinophils may be recruited into the lymphoma not only by HRS cells but also by fibroblasts through secretion of eotaxin and CCL5. CCL5 secreted by fibroblasts may also contribute to the attraction of CD4+ T cells (not shown). Eosinophils and mast cells may stimulate HRS cells through CD30CD30 ligand (CD30L) interaction, whereas granulocytes may stimulate HRS cells through APRILBCMA interaction and the secretion of nerve growth factor (NGF), which binds to the receptor tyrosine kinase TRKA on HRS cells. The cellular interactions between CD4+ TH cells probably involve adhesion molecules (CD54CD18 or CD54CD11A) and key molecules of cognate B cellT cell interaction, that is, CD40CD40L, major histocompatibility complex (MHC) class IITCR (T-cell receptor) and CD80CD28. Cytotoxic T (TC) cells are inhibited through TReg cells by secretion of IL-10 and perhaps additional mechanisms, and directly by the HRS cells though secretion of immunosuppressive mediators and expression of the programmed cell death 1 ligand (PD1L) by HRS cells. Note that Hodgkins lymphoma cases show wide variation in the frequencies of cells shown. TGF, transforming growth factor-; TNF, tumour necrosis factor-.

TH2 response

A T-helper-2-cell-mediated immune response involves the production of cytokines, such as IL-4, that stimulate antibody production. TH2 cytokines promote secretory immune responses of mucosal surfaces to extracellular pathogens and allergic reactions.

lymphoma microenvironment is presumably established not only by the chemokine-mediated attraction of such cells, as discussed above, but also by induction of differentiation of naive CD4+ T cells into TReg cells by HRS cells148. unexpectedly, high numbers of TReg cells in the Hodgkins lymphoma microenvironment have been linked to a good prognosis, indicating that TReg cells may also have some suppressive activity on HRS cells or on other inflammatory cells that support HRS cell survival and/or proliferation149,150.

HRS cells may further modulate their cellular microenvironment by shifting the TH response from an anticellular TH1 response to a humoral TH2 response, which often has tumour-promoting activities 151. HRS cells also produce the immunosuppressive cytokines IL-10 and TGF, and galectin 1 and prostaglandin e2, which inhibit T-cell effector functions106,137,152154. Moreover, T-cell effector functions are inhibited by binding of programmed cell death protein 1 (PD1) on T cells to the PD1 ligand that is expressed by HRS cells155,156.
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Infectious mononucleosis
A self-limiting disease usually caused by a delayed primary infection with eBV in adolescents or adults.

In several tumours, including some B-cell lymphomas, chronic inflammatory responses against infectious agents or resulting from autoimmune diseases are thought to be initiating events for tumour development151. However, there is little evidence to date for a close association of Hodgkins lymphoma development with autoimmune diseases or chronic infections. Although patients with infectious mononucleosis have an increased risk of developing eBV+ Hodgkins lymphoma157, in this instance Hodgkins lymphoma usually develops several years after the resolution of the primary eBV infection, and the HRS-like cells that are observed during infectious mononucleosis lack key features of HRS cells158,159. Thus, it seems to be more likely that in Hodgkins lymphoma the typical inflammatory response usually develops along with the generation of the malignant HRS cells.

Conclusions and perspective Classical Hodgkins lymphoma (and, in some features, also NLPHL) is a unique malignancy owing to several key characteristics. First, no other B-lymphoid malignancy shows such a dramatic loss of the B-cell phenotype other lymphomas usually retain many typical features of their cell of origin. Although several factors that contribute to this reprogramming have been identified (fIG. 2), the events that initiate this process are still unclear. It remains to be resolved whether the reprogramming occurs as a byproduct of some as yet unidentified transforming events, or whether it is a prerequisite for the survival of the HRS precursor cells the crippled germinal centre B cells. Second, although deregulation of signalling pathways is a key feature of all lymphomas, the constitutive deregulated activation of multiple signalling pathways in Hodgkins lymphoma is not evident in other lymphoid malignancies. HRS cells show not only constitutive activity of signalling pathways that are normally only transiently activated in B cells (such as NF-B, jakStat and PI3K), but also activation of signalling molecules and pathways that are not normally activated in B cells (such as notch 1 and multiple receptor tyrosine kinases). Third, although cellular interactions with non-tumour cells are important in other lymphomas31, the extent of the cellular infiltration, involving many different types of cells, and the resemblance of the microenvironment to an inflammatory

response is unusual in Hodgkins lymphoma. A picture is emerging of HRS cells acting as master regulators of the chronic inflammatory response and of the multiple and diverse signalling pathways that are activated, orchestrating a microenvironment that promotes HRS cell growth and impairs the immune surveillance against these cells. As signalling molecules not normally expressed by B cells contribute to this regulation of the microenvironment, the reprogramming of HRS cells may be of selective advantage for the HRS cells also in this regard. The recent identification of pathogenetic mechanisms in HRS cells and essential cellular interactions offers new approaches for targeted therapy that would reduce the toxicity of current treatment regimens160. Clinically, it would also be important to identify the ~10% of patients who are not cured by standard therapy. There is an urgent need for identification of biological markers that distinguish, at the time of diagnosis, primary progressive cases of Hodgkins lymphoma from cases responding well to therapy. An exciting prospect for the identification of further genetic lesions in HRS and L&H cells lies in the application of modern global microarray-based approaches to detect genomic gains and losses161 and the application of massively parallel sequencing to detect mutations in oncogenes and tumour suppressor genes, once the technical obstacles to apply these methods to the rare HRS and L&H cells have been overcome. Massively parallel sequencing of the genome or transcriptome of HRS cells also offers the possibility to clarify whether another virus besides eBV is involved in Hodgkins lymphoma pathogenesis. In particular, young adult cases of classical Hodgkins lymphoma, which are rarely associated with eBV, show epidemiological features that suggest involvement of an infectious agent, presumably a virus162, but no such virus has been identified so far. Large-scale sequencing projects may finally reveal whether such an infectious agent can exist in HRS cells. Given the importance of epigenetic alterations in tumour cells, it will also be essential to further our understanding of such alterations in HRS and L&H cells. Finally, we are also beginning to understand that deregulated expression of microRNAs may contribute163,164, but further studies are needed to clarify which roles specific microRNAs have in the pathogenesis of Hodgkins lymphoma.

1. 2.

3.

4.

5. 6.

Hodgkin, T. On some morbid appearances of the absorbent glands and spleen. Med. Chir. Trans. 17, 68114 (1832). Reed, D. On the pathological changes in Hodgkins disease with special reference to its relation to tuberculosis. John Hopkins Hosp. Rep. 10, 133193 (1902). Sternberg, C. be r eine eigenartige unter dem Bilde der Pseudoleukmie verlaufende Tuberkolose des lymphatischen Apparates. Z. Heilkunde 19, 2190 (1898) (in German). Jaffe, E. S., Harris, N. L., Stein, H. & Vardiman, J. W. WHO Classification of Tumors: Pathology and Genetics of Tumors of Haematopoietic and Lymphoid Tissues (IARC Press, Lyon, 2001). Diehl, V., Thomas, R. K. & Re, D. Part II: Hodgkins lymphoma diagnosis and treatment. Lancet Oncol. 5, 1926 (2004). Rajewsky, K. Clonal selection and learning in the antibody system. Nature 381, 751758 (1996).

Kppers, R., Zhao, M., Hansmann, M. L. & Rajewsky, K. Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO J. 12, 49554967 (1993). 8. Allen, C. D., Okada, T. & Cyster, J. G. Germinal-center organization and cellular dynamics. Immunity 27, 190202 (2007). 9. Carbone, A. et al. Expression status of BCL-6 and syndecan-1 identifies distinct histogenetic subtypes of Hodgkins disease. Blood 92, 22202228 (1998). 10. Greiner, A. et al. Differential expression of activation-induced cytidine deaminase (AID) in nodular lymphocyte-predominant and classical Hodgkin lymphoma. J. Pathol. 205, 541547 (2005). 11. Hansmann, M.-L., Weiss, L. M., Stein, H., Harris, N. L. & Jaffe, E. S. in Hodgkins Disease (eds Mauch, P. M., Armitage, J. O., Diehl, V., Hoppe, R. T. & Weiss, L. M.) 7.

169180 (Lippencott Williams & Wilkins, Philadelphia, 1999). 12. Braeuninger, A. et al. Hodgkin and ReedSternberg cells in lymphocyte predominant Hodgkin disease represent clonal populations of germinal centerderived tumor B cells. Proc. Natl Acad. Sci. USA 94, 93379342 (1997). 13. Kppers, R. et al. Hodgkin disease: Hodgkin and ReedSternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development. Proc. Natl Acad. Sci. USA 91, 1096210966 (1994). The first demonstration that HRS and L&H cells are clonal populations of B cells. 14. Marafioti, T. et al. Origin of nodular lymphocytepredominant Hodgkins disease from a clonal expansion of highly mutated germinal-center B cells. N. Engl. J. Med. 337, 453458 (1997).

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REVIEWS
15. Brune, V. et al. Origin and pathogenesis of nodular lymphocyte-predominant Hodgkin lymphoma as revealed by global gene expression analysis. J. Exp. Med. 205, 22512268 (2008). 16. Kanzler, H., Kppers, R., Hansmann, M. L. & Rajewsky, K. Hodgkin and ReedSternberg cells in Hodgkins disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells. J. Exp. Med. 184, 14951505 (1996). This study provided the first evidence that HRS cells are derived from crippled, BCR-deficient germinal centre B cells. 17. Bruninger, A., Wacker, H. H., Rajewsky, K., Kppers, R. & Hansmann, M. L. Typing the histogenetic origin of the tumor cells of lymphocyte-rich classical Hodgkins lymphoma in relation to tumor cells of classical and lymphocyte-predominance Hodgkins lymphoma. Cancer Res. 63, 16441651 (2003). 18. Kppers, R. & Rajewsky, K. The origin of Hodgkin and Reed/Sternberg cells in Hodgkins disease. Annu. Rev. Immunol. 16, 471493 (1998). 19. Klein, U. et al. Somatic hypermutation in normal and transformed human B cells. Immunol. Rev. 162, 261280 (1998). 20. Irsch, J. et al. Class switch recombination was specifically targeted to immunoglobulin (Ig)G4 or IgA in Hodgkins disease-derived cell lines. Br. J. Haematol. 113, 785793 (2001). 21. Martin-Subero, J. I. et al. Chromosomal breakpoints affecting immunoglobulin loci are recurrent in Hodgkin and ReedSternberg cells of classical Hodgkin lymphoma. Cancer Res. 66, 1033210338 (2006). 22. Bruninger, A. et al. Identification of common germinal-center B-cell precursors in two patients with both Hodgkins disease and Non-Hodgkins lymphoma. N. Engl. J. Med. 340, 12391247 (1999). 23. Marafioti, T. et al. Classical Hodgkins disease and follicular lymphoma originating from the same germinal center B cell. J. Clin. Oncol. 17, 38043809 (1999). References 22 and 23 demonstrated the close relationship of classical Hodgkins lymphoma and non-Hodgkins lymphomas by identifying common germinal centre B-cell precursors in composite lymphomas. 24. Mschen, M. et al. Rare occurrence of classical Hodgkins disease as a T cell lymphoma. J. Exp. Med. 191, 387394 (2000). 25. Seitz, V. et al. Detection of clonal T-cell receptor gamma-chain gene rearrangements in Reed Sternberg cells of classic Hodgkin disease. Blood 95, 30203024 (2000). 26. Tzankov, A. et al. Rare expression of T-cell markers in classical Hodgkins lymphoma. Mod. Pathol. 18, 15421549 (2005). 27. Aguilera, N. S. et al. Gene rearrangement and comparative genomic hybridization studies of classic Hodgkin lymphoma expressing T-cell antigens. Arch. Pathol. Lab. Med. 130, 17721779 (2006). 28. Willenbrock, K. et al. T-cell variant of classical Hodgkins lymphoma with nodal and cutaneous manifestations demonstrated by single-cell polymerase chain reaction. Lab. Invest. 82, 11031109 (2002). 29. Barry, T. S., Jaffe, E. S., Sorbara, L., Raffeld, M. & Pittaluga, S. Peripheral T-cell lymphomas expressing CD30 and CD15. Am. J. Surg. Pathol. 27, 15131522 (2003). 30. Willenbrock, K. et al. Common features and differences in the transcriptome of large cell anaplastic lymphoma and classical Hodgkins lymphoma. Haematologica 91, 596604 (2006). 31. Kppers, R. Mechanisms of B-cell lymphoma pathogenesis. Nature Rev. Cancer 5, 251262 (2005). 32. Schwering, I. et al. Loss of the B-lineage-specific gene expression program in Hodgkin and ReedSternberg cells of Hodgkin lymphoma. Blood 101, 15051512 (2003). This study demonstrated the global downregulation of B-cell molecules in HRS cells. 33. Carbone, A. et al. Expression of functional CD40 antigen on ReedSternberg cells and Hodgkins disease cell lines. Blood 85, 780789 (1995). 34. Van Gool, S. W. et al. Expression of B72 (CD86) molecules by ReedSternberg cells of Hodgkins disease. Leukemia 11, 846851 (1997). 35. Mathas, S. et al. Intrinsic inhibition of transcription factor E2A by HLH proteins ABF-1 and Id2 mediates reprogramming of neoplastic B cells in Hodgkin lymphoma. Nature Immunol. 7, 207215 (2006). This paper provided insight into the mechanisms involved in the reprogramming of HRS cells. 36. Takahashi, H. et al. Immunophenotypes of Reed Sternberg cells and their variants: a study of 68 cases of Hodgkins disease. Tohoku J. Exp. Med. 177, 193211 (1995). 37. Drexler, H. G. Recent results on the biology of Hodgkin and ReedSternberg cells. I. Biopsy material. Leuk. Lymphoma 8, 283313 (1992). 38. van den Berg, A., Visser, L. & Poppema, S. High expression of the CC chemokine TARC in Reed Sternberg cells. A possible explanation for the characteristic T-cell infiltration Hodgkins lymphoma. Am. J. Pathol. 154, 16851691 (1999). 39. Re, D. et al. Oct-2 and Bob-1 deficiency in Hodgkin and Reed Sternberg cells. Cancer Res. 61, 20802084 (2001). 40. Stein, H. et al. Down-regulation of BOB.1/OBF.1 and Oct2 in classical Hodgkin disease but not in lymphocyte predominant Hodgkin disease correlates with immunoglobulin transcription. Blood 97, 496501 (2001). 41. Torlakovic, E., Tierens, A., Dang, H. D. & Delabie, J. The transcription factor PU.1, necessary for B-cell development is expressed in lymphocyte predominance, but not classical Hodgkins disease. Am. J. Pathol. 159, 18071814 (2001). 42. Doerr, J. R. et al. Patterned CpG methylation of silenced B cell gene promoters in classical Hodgkin lymphoma-derived and primary effusion lymphoma cell lines. J. Mol. Biol. 350, 631640 (2005). 43. Ushmorov, A. et al. Epigenetic processes play a major role in B-cell-specific gene silencing in classical Hodgkin lymphoma. Blood 107, 24932500 (2005). 44. Hertel, C. B., Zhou, X. G., Hamilton-Dutoit, S. J. & Junker, S. Loss of B cell identity correlates with loss of B cell-specific transcription factors in Hodgkin/Reed Sternberg cells of classical Hodgkin lymphoma. Oncogene 21, 49084920 (2002). 45. Pongubala, J. M. et al. Transcription factor EBF restricts alternative lineage options and promotes B cell fate commitment independently of Pax5. Nature Immunol. 9, 203215 (2008). 46. Kppers, R. et al. Identification of Hodgkin and Reed Sternberg cell-specific genes by gene expression profiling. J. Clin. Invest. 111, 529537 (2003). 47. Renn, C. et al. Aberrant expression of ID2, a suppressor of B-cell-specific gene expression, in Hodgkins lymphoma. Am. J. Pathol. 169, 655664 (2006). 48. Hacker, C. et al. Transcriptional profiling identifies Id2 function in dendritic cell development. Nature Immunol. 4, 380386 (2003). 49. Yokota, Y. et al. Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2. Nature 397, 702706 (1999). 50. Foss, H. D. et al. Frequent expression of the B-cell-specific activator protein in ReedSternberg cells of classical Hodgkins disease provides further evidence for its B-cell origin. Blood 94, 31083113 (1999). 51. Cobaleda, C., Schebesta, A., Delogu, A. & Busslinger, M. Pax5: the guardian of B cell identity and function. Nature Immunol. 8, 463470 (2007). 52. Jundt, F. et al. Activated Notch 1 signaling promotes tumor cell proliferation and survival in Hodgkin and anaplastic large cell lymphoma. Blood 99, 33983403 (2001). 53. Jundt, F. et al. Aberrant expression of Notch1 interferes with the B-lymphoid phenotype of neoplastic B cells in classical Hodgkin lymphoma. Leukemia 22 15871594 (2008). References 52 and 53 showed aberrant expression of notch 1 in HRS cells and that this contributes to the lost B-cell phenotype of these cells. 54. Scheeren, F. A. et al. IL-21 is expressed in Hodgkin lymphoma and activates STAT5; evidence that activated STAT5 is required for Hodgkin lymphomagenesis. Blood 111, 47064715 (2008). 55. Schneider, E. M. et al. The early transcription factor GATA-2 is expressed in classical Hodgkins lymphoma. J. Pathol. 204, 538545 (2004). 56. Kumano, K. et al. Notch1 inhibits differentiation of hematopoietic cells by sustaining GATA-2 expression. Blood 98, 32833289 (2001). 57. Dukers, D. F. et al. Unique polycomb gene expression pattern in Hodgkins lymphoma and Hodgkins lymphoma-derived cell lines. Am. J. Pathol. 164, 873881 (2004). 58. Raaphorst, F. M. et al. Coexpression of BMI-1 and EZH2 polycomb group genes in ReedSternberg cells of Hodgkins disease. Am. J. Pathol. 157, 709715 (2000). 59. Sanchez-Beato, M. et al. Abnormal PcG protein expression in Hodgkins lymphoma. Relation with E2F6 and NFkappaB transcription factors. J. Pathol. 204, 528537 (2004). 60. Hu, M. et al. Multilineage gene expression precedes commitment in the hemopoietic system. Genes Dev. 11, 774785 (1997). 61. Miyamoto, T. et al. Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment. Dev. Cell 3, 137147 (2002). 62. Dutton, A. et al. Bmi-1 is induced by the EpsteinBarr virus oncogene LMP1 and regulates the expression of viral target genes in Hodgkin lymphoma cells. Blood 109, 25972603 (2007). 63. Feuerborn, A. et al. Dysfunctional p53 deletion mutants in cell lines derived from Hodgkins lymphoma. Leuk. Lymphoma 47, 19321940 (2006). 64. Maggio, E. M., Stekelenburg, E., Van den Berg, A. & Poppema, S. TP53 gene mutations in Hodgkin lymphoma are infrequent and not associated with absence of EpsteinBarr virus. Int. J. Cancer 94, 6066 (2001). 65. Montesinos-Rongen, M., Roers, A., Kppers, R., Rajewsky, K. & Hansmann, M.-L. Mutation of the p53 gene is not a typical feature of Hodgkin and Reed Sternberg cells in Hodgkins disease. Blood 94, 17551760 (1999). 66. Kashkar, H. et al. XIAP-mediated caspase inhibition in Hodgkins lymphoma-derived B cells. J. Exp. Med. 198, 341347 (2003). 67. Dutton, A. et al. Expression of the cellular FLICEinhibitory protein (c-FLIP) protects Hodgkins lymphoma cells from autonomous Fas-mediated death. Proc. Natl Acad. Sci. USA 101, 66116616 (2004). 68. Mathas, S. et al. c-FLIP mediates resistance of Hodgkin/ReedSternberg cells to death receptorinduced apoptosis. J. Exp. Med. 199, 10411052 (2004). 69. Wlodarska, I. et al. Frequent occurrence of BCL6 rearrangements in nodular lymphocyte predominance Hodgkin lymphoma but not in classical Hodgkin lymphoma. Blood 101, 706710 (2003). 70. Joos, S. et al. Genomic imbalances including amplification of the tyrosine kinase gene JAK2 in CD30+ Hodgkin cells. Cancer Res. 60, 549552 (2000). 71. Weniger, M. A. et al. Mutations of the tumor suppressor gene SOCS-1 in classical Hodgkin lymphoma are frequent and associated with nuclear phospho-STAT5 accumulation. Oncogene 25, 26792684 (2006). This paper showed that frequent somatic mutations in SOCS1 in HRS cells were probably a factor contributing to constitutive STAT activity in these tumour cells. 72. Mottok, A., Renn, C., Willenbrock, K., Hansmann, M. L. & Bruninger, A. Somatic hypermutation of SOCS1 in lymphocyte-predominant Hodgkin lymphoma is accompanied by high JAK2 expression and activation of STAT6. Blood 110, 33873390 (2007). 73. Barth, T. F. et al. Gains of 2p involving the REL locus correlate with nuclear c-Rel protein accumulation in neoplastic cells of classical Hodgkin lymphoma. Blood 101, 36813686 (2003). 74. Joos, S. et al. Classical Hodgkin lymphoma is characterized by recurrent copy number gains of the short arm of chromosome 2. Blood 99, 13811387 (2002). 75. Martin-Subero, J. I. et al. Recurrent involvement of the REL and BCL11A loci in classical Hodgkin lymphoma. Blood 99, 14741477 (2002). 76. Martin-Subero, J. I. et al. Chromosomal rearrangements involving the BCL3 locus are recurrent in classical Hodgkin and peripheral T-cell lymphoma. Blood 108, 401402 (2006). 77. Mathas, S. et al. Elevated NF-B p50 complex formation and Bcl-3 expression in classical Hodgkin, anaplastic large-cell, and other peripheral T-cell lymphomas. Blood 106, 42874293 (2005). 78. Cabannes, E., Khan, G., Aillet, F., Jarrett, R. F. & Hay, R. T. Mutations in the IB gene in Hodgkins disease suggest a tumour suppressor role for IB. Oncogene 18, 30633070 (1999). 79. Emmerich, F. et al. Overexpression of I kappa B alpha without inhibition of NF-B activity and mutations in the I kappa B alpha gene in ReedSternberg cells. Blood 94, 31293134 (1999).

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80. Jungnickel, B. et al. Clonal deleterious mutations in the IB gene in the malignant cells in Hodgkins disease. J. Exp. Med. 191, 395401 (2000). References 7880 established somatic mutations in NFKBIA in a fraction of Hodgkins lymphoma cases as a cause for constitutive NF-B activity in HRS cells. 81. Emmerich, F. et al. Inactivating I kappa B epsilon mutations in Hodgkin/ReedSternberg cells. J. Pathol. 201, 413420 (2003). 82. Baus, D. & Pfitzner, E. Specific function of STAT3, SOCS1, and SOCS3 in the regulation of proliferation and survival of classical Hodgkin lymphoma cells. Int. J. Cancer 118, 14041413 (2006). 83. Kube, D. et al. STAT3 is constitutively activated in Hodgkin cell lines. Blood 98, 762770 (2001). 84. Skinnider, B. F. et al. Signal transducer and activator of transcription 6 is frequently activated in Hodgkin and ReedSternberg cells of Hodgkin lymphoma. Blood 99, 618626 (2002). 85. Kapp, U. et al. Interleukin 13 is secreted by and stimulates the growth of Hodgkin and ReedSternberg cells. J. Exp. Med. 189, 19391946 (1999). 86. Hinz, M. et al. Nuclear factor B-dependent gene expression profiling of Hodgkins disease tumor cells, pathogenetic significance, and link to constitutive signal transducer and activator of transcription 5a activity. J. Exp. Med. 196, 605617 (2002). 87. Lamprecht, B. et al. Aberrant expression of the Th2 cytokine IL-21 in Hodgkin lymphoma cells regulates STAT3 signaling and attracts Treg cells via regulation of MIP-3. Blood 112, 33393347 (2008). 88. Chiu, A. et al. Hodgkin lymphoma cells express TACI and BCMA receptors and generate survival and proliferation signals in response to BAFF and APRIL. Blood 109, 729739 (2007). 89. Fiumara, P. et al. Functional expression of receptor activator of nuclear factor B in Hodgkin disease cell lines. Blood 98, 27842790 (2001). 90. Schwab, U. et al. Production of a monoclonal antibody specific for Hodgkin and SternbergReed cells of Hodgkins disease and a subset of normal lymphoid cells. Nature 299, 6567 (1982). 91. Carbone, A., Gloghini, A., Gruss, H. J. & Pinto, A. CD40 ligand is constitutively expressed in a subset of T cell lymphomas and on the microenvironmental reactive T cells of follicular lymphomas and Hodgkins disease. Am. J. Pathol. 147, 912922 (1995). 92. Molin, D. et al. Mast cells express functional CD30 ligand and are the predominant CD30L-positive cells in Hodgkins disease. Br. J. Haematol. 114, 616623 (2001). 93. Pinto, A. et al. Human eosinophils express functional CD30 ligand and stimulate proliferation of a Hodgkins disease cell line. Blood 88, 32993305 (1996). 94. Hirsch, B. et al. CD30-induced signaling is absent in Hodgkins cells but present in anaplastic large cell lymphoma cells. Am. J. Pathol. 172, 510520 (2008). 95. Horie, R. et al. Ligand-independent signaling by overexpressed CD30 drives NF-B activation in HodgkinReedSternberg cells. Oncogene 21, 24932503 (2002). 96. Nishikori, M., Ohno, H., Haga, H. & Uchiyama, T. Stimulation of CD30 in anaplastic large cell lymphoma leads to production of nuclear factor-B p52, which is associated with hyperphosphorylated Bcl-3. Cancer Sci. 96, 487497 (2005). 97. Schwaller, J. et al. Paracrine promotion of tumor development by the TNF ligand APRIL in Hodgkins disease. Leukemia 21, 13241327 (2007). 98. Cheng, P. et al. Notch-1 regulates NF-B activity in hemopoietic progenitor cells. J. Immunol. 167, 44584467 (2001). 99. Rodig, S. J. et al. TRAF1 expression and c-Rel activation are useful adjuncts in distinguishing classical Hodgkin lymphoma from a subset of morphologically or immunophenotypically similar lymphomas. Am. J. Surg. Pathol. 29, 196203 (2005). 100. Dutton, A., Reynolds, G. M., Dawson, C. W., Young, L. S. & Murray, P. G. Constitutive activation of phosphatidyl-inositide 3 kinase contributes to the survival of Hodgkins lymphoma cells through a mechanism involving Akt kinase and mTOR. J. Pathol. 205, 498506 (2005). 101. Georgakis, G. V. et al. Inhibition of the phosphatidylinositol-3 kinase/Akt promotes G1 cell cycle arrest and apoptosis in Hodgkin lymphoma. Br. J. Haematol. 132, 503511 (2006). 102. Mathas, S. et al. Aberrantly expressed c-Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF-kappa B. EMBO J. 21, 41044113 (2002). 103. Nagel, S. et al. Comprehensive analysis of homeobox genes in Hodgkin lymphoma cell lines identifies dysregulated expression of HOXB9 mediated via ERK5 signaling and BMI1. Blood 109, 30153023 (2007). 104. Renn, C., Willenbrock, K., Kppers, R., Hansmann, M.-L. & Bruninger, A. Autocrine and paracrine activated receptor tyrosine kinases in classical Hodgkin lymphoma. Blood 105, 40514059 (2005). 105. Zheng, B. et al. MEK/ERK pathway is aberrantly active in Hodgkin disease: a signaling pathway shared by CD30, CD40, and RANK that regulates cell proliferation and survival. Blood 102, 10191027 (2003). 106. Juszczynski, P. et al. The AP1-dependent secretion of galectin-1 by Reed Sternberg cells fosters immune privilege in classical Hodgkin lymphoma. Proc. Natl Acad. Sci. USA 104, 1313413139 (2007). 107. Watanabe, M. et al. AP-1 mediated relief of repressive activity of the CD30 promoter microsatellite in Hodgkin and ReedSternberg cells. Am. J. Pathol. 163, 633641 (2003). 108. Renn, C. et al. High expression of several tyrosine kinases and activation of the PI3K/AKT pathway in mediastinal large B cell lymphoma reveals further similarities to Hodgkin lymphoma. Leukemia 21, 780787 (2007). 109. Schmitz, R., Stanelle, J., Hansmann, M.-L. & Kppers, R. Pathogenesis of classical and lymphocytepredominant Hodgkin lymphoma. Annu. Rev. Pathol. (in the press). 110. Anagnostopoulos, I. et al. European Task Force on Lymphoma project on lymphocyte predominance Hodgkin disease: histologic and immunohistologic analysis of submitted cases reveals 2 types of Hodgkin disease with a nodular growth pattern and abundant lymphocytes. Blood 96, 18891899 (2000). 111. Chang, K. C., Khen, N. T., Jones, D. & Su, I. J. Epstein Barr virus is associated with all histological subtypes of Hodgkin lymphoma in Vietnamese children with special emphasis on the entity of lymphocyte predominance subtype. Hum. Pathol. 36, 747755 (2005). 112. Dolcetti, R., Boiocchi, M., Gloghini, A. & Carbone, A. Pathogenetic and histogenetic features of HIVassociated Hodgkins disease. Eur. J. Cancer 37, 12761287 (2001). 113. Kutok, J. L. & Wang, F. Spectrum of EpsteinBarr virus-associated diseases. Annu. Rev. Pathol. 1, 375404 (2006). 114. Baumforth, K. R. et al. Expression of the EpsteinBarr virus-encoded EpsteinBarr virus nuclear antigen 1 in Hodgkins lymphoma cells mediates up-regulation of CCL20 and the migration of regulatory T cells. Am. J. Pathol. 173, 195204 (2008). 115. Flavell, J. R. et al. Down-regulation of the TGF- target gene, PTPRK, by the EpsteinBarr virus encoded EBNA1 contributes to the growth and survival of Hodgkin lymphoma cells. Blood 111, 292301 (2008). 116. Kulwichit, W. et al. Expression of the EpsteinBarr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice. Proc. Natl Acad. Sci. USA 95, 1196311968 (1998). 117. Kilger, E., Kieser, A., Baumann, M. & Hammerschmidt, W. EpsteinBarr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor. EMBO J. 17, 17001709 (1998). 118. Young, L. S. & Murray, P. G. EpsteinBarr virus and oncogenesis: from latent genes to tumours. Oncogene 22, 51085121 (2003). 119. Alber, G. et al. Molecular mimicry of the antigen receptor signalling motif by transmembrane proteins of the EpsteinBarr virus and the bovine leukemia virus. Curr. Biol. 3, 333339 (1993). 120. Caldwell, R. G., Wilson, J. B., Anderson, S. J. & Longnecker, R. EpsteinBarr virus LMP2A drives B cell development and survival in the absence of normal B cell receptor signals. Immunity 9, 405411 (1998). 121. Liu, Y. J. et al. Mechanism of antigen-driven selection in germinal centres. Nature 342, 929931 (1989). 122. Portis, T., Dyck, P. & Longnecker, R. EpsteinBarr Virus (EBV) LMP2A induces alterations in gene transcription similar to those observed in Reed Sternberg cells of Hodgkin lymphoma. Blood 102, 41664178 (2003). 123. Vockerodt, M. et al. The EpsteinBarr virus oncoprotein, latent membrane protein-1, reprograms germinal centre B cells towards a Hodgkins Reed Sternberg-like phenotype. J. Pathol. 216, 8392 (2008). 124. Renn, C. et al. The aberrant coexpression of several receptor tyrosine kinases is largely restricted to EBV-negative cases of classical Hodgkins lymphoma. Int. J. Cancer 120, 25042509 (2007). 125. Bechtel, D., Kurth, J., Unkel, C. & Kppers, R. Transformation of BCR-deficient germinal-center B cells by EBV supports a major role of the virus in the pathogenesis of Hodgkin and posttransplantation lymphomas. Blood 106, 43454350 (2005). 126. Mancao, C., Altmann, M., Jungnickel, B. & Hammerschmidt, W. Rescue of crippled germinal center B cells from apoptosis by EpsteinBarr virus. Blood 106, 43394344 (2005). 127. Chaganti, S. et al. EpsteinBarr virus infection in vitro can resue germinal centre B cells with inactivated immunoglobulin genes. Blood 106, 42494252 (2005). 128. Mancao, C. & Hammerschmidt, W. EpsteinBarr virus latent membrane protein 2A is a B-cell receptor mimic and essential for B-cell survival. Blood 110, 37153721 (2007). 129. Bruninger, A. et al. Molecular biology of Hodgkin and Reed/Sternberg cells in Hodgkins lymphoma. Int. J. Cancer 118, 18531861 (2006). 130. Ward, R. J. & Dirks, P. B. Cancer stem cells: at the headwaters of tumor development. Annu. Rev. Pathol. 2, 175189 (2007). 131. Drexler, H. G., Gignac, S. M., Hoffbrand, A. V. & Minowada, J. Formation of multinucleated cells in a Hodgkins-disease-derived cell line. Int. J. Cancer 43, 10831090 (1989). 132. Newcom, S. R., Kadin, M. E. & Phillips, C. L-428 ReedSternberg cells and mononuclear Hodgkins cells arise from a single cloned mononuclear cell. Int. J. Cell Cloning 6, 417431 (1988). 133. Jansen, M. P. et al. Morphologically normal, CD30negative B-lymphocytes with chromosome aberrations in classical Hodgkins disease: the progenitor cell of the malignant clone? J. Pathol. 189, 527532 (1999). 134. Barrios, L. et al. Chromosome abnormalities in peripheral blood lymphocytes from untreated Hodgkins patients. A possible evidence for chromosome instability. Hum. Genet. 78, 320324 (1988). 135. MKacher, R. et al. Baseline and treatment-induced chromosomal abnormalities in peripheral blood lymphocytes of Hodgkins lymphoma patients. Int. J. Radiat. Oncol. Biol. Phys. 57, 321326 (2003). 136. Spieker, T. et al. Molecular single-cell analysis of the clonal relationship of small EpsteinBarr virus-infected cells and EpsteinBarr virus-harboring Hodgkin and Reed/Sternberg cells in Hodgkin disease. Blood 96, 31333138 (2000). 137. Skinnider, B. F. & Mak, T. W. The role of cytokines in classical Hodgkin lymphoma. Blood 99, 42834297 (2002). 138. Aldinucci, D. et al. Expression of CCR5 receptors on ReedSternberg cells and Hodgkin lymphoma cell lines: involvement of CCL5/Rantes in tumor cell growth and microenvironmental interactions. Int. J. Cancer 122, 769776 (2008). 139. Fischer, M. et al. Expression of CCL5/RANTES by Hodgkin and ReedSternberg cells and its possible role in the recruitment of mast cells into lymphomatous tissue. Int. J. Cancer 107, 197201 (2003). 140. Kapp, U. et al. Disseminated growth of Hodgkinsderived cell lines L540 and L540cy in immune-deficient SCID mice. Ann. Oncol. 5 (Suppl. 1),121126 (1994). 141. Biggar, R. J. et al. Hodgkin lymphoma and immunodeficiency in persons with HIV/AIDS. Blood 108, 37863791 (2006). 142. Aldinucci, D. & Gattei, V. Interleukin-3 receptors in Hodgkins disease. Am. J. Pathol. 162, 356357 (2003). 143. Aldinucci, D. et al. Expression of functional interleukin-3 receptors on Hodgkin and Reed Sternberg cells. Am. J. Pathol. 160, 585596 (2002). 144. Aldinucci, D., Lorenzon, D., Olivo, K., Rapana, B. & Gattei, V. Interactions between tissue fibroblasts in lymph nodes and Hodgkin/ReedSternberg cells. Leuk. Lymphoma 45, 17311739 (2004). 145. Jundt, F. et al. Hodgkin/ReedSternberg cells induce fibroblasts to secrete eotaxin, a potent chemoattractant for T cells and eosinophils. Blood 94, 20652071 (1999). 146. Gandhi, M. K. et al. Expression of LAG-3 by tumorinfiltrating lymphocytes is coincident with the suppression of latent membrane antigen-specific CD8+ T-cell function in Hodgkin lymphoma patients. Blood 108, 22802289 (2006). References 106 and 146 showed that secretion of galectin 1 by HRS cells is involved in the suppression of cytotoxic T-cell responses against the HRS cells.

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147. Marshall, N. A. et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma. Blood 103, 17551762 (2004). This article established that regulatory T cells are frequently found in the Hodgkins lymphoma microenvironment. 148. Tanijiri, T. et al. Hodgkins ReedSternberg cell line (KM-H2) promotes a bidirectional differentiation of CD4+CD25+Foxp3+ T cells and CD4+ cytotoxic T lymphocytes from CD4+ naive T cells. J. Leukoc. Biol. 82, 576584 (2007). 149. Alvaro, T. et al. Outcome in Hodgkins lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells. Clin. Cancer Res. 11, 14671473 (2005). 150. Kelley, T. W., Pohlman, B., Elson, P. & Hsi, E. D. The ratio of FOXP3+ regulatory T cells to granzyme B+ cytotoxic T/NK cells predicts prognosis in classical Hodgkin lymphoma and is independent of bcl-2 and MAL expression. Am. J. Clin. Pathol. 128, 958965 (2007). 151. Tan, T. T. & Coussens, L. M. Humoral immunity, inflammation and cancer. Curr. Opin. Immunol. 19, 209216 (2007). 152. Gandhi, M. K. et al. Galectin-1 mediated suppression of EpsteinBarr virus specific T-cell immunity in classic Hodgkin lymphoma. Blood 110, 13261329 (2007). 153. Newcom, S. R. & Gu, L. Transforming growth factor 1 messenger RNA in ReedSternberg cells in nodular sclerosing Hodgkins disease. J. Clin. Pathol. 48, 160163 (1995). 154. Chemnitz, J. M. et al. Prostaglandin E2 impairs CD4+ T cell activation by inhibition of lck: implications in Hodgkins lymphoma. Cancer Res. 66, 11141122 (2006). 155. Chemnitz, J. M. et al. RNA fingerprints provide direct evidence for the inhibitory role of TGF and PD-1 on CD4+ T cells in Hodgkin lymphoma. Blood 110, 32263233 (2007). 156. Yamamoto, R. et al. PD-1PD-1 ligand interaction contributes to immunosuppressive microenvironment of Hodgkin lymphoma. Blood 111, 32203224 (2008). 157. Hjalgrim, H. et al. Infectious mononucleosis, childhood social environment, and risk of Hodgkin lymphoma. Cancer Res. 67, 23822388 (2007). 158. Kurth, J., Hansmann, M.-L., Rajewsky, K. & Kppers, R. EpsteinBarr virus-infected B cells expanding in germinal centers of infectious mononucleosis patients do not participate in the germinal center reaction. Proc. Natl Acad. Sci. USA 100, 47304735 (2003). 159. Kurth, J. et al. EBV-infected B cells in infectious mononucleosis: viral strategies for spreading in the B cell compartment and establishing latency. Immunity 13, 485495 (2000). 160. Buglio, D. et al. Vorinostat inhibits STAT6-mediated TH2 cytokine and TARC production and induces cell death in Hodgkin lymphoma cell lines. Blood 112, 14241433 (2008). 161. Hartmann, S. et al. Detection of genomic imbalances in microdissected Hodgkin and ReedSternberg cells of classical Hodgkins lymphoma by array-based comparative genomic hybridization. Haematologica 93, 13181326 (2008). 162. Gutensohn, N. & Cole, P. Epidemiology of Hodgkins disease. Semin. Oncol. 7, 92102 (1980). 163. Kluiver, J. et al. BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas. J. Pathol. 207, 243249 (2005). 164. Nie, K. et al. MicroRNA-mediated down-regulation of PRDM1/Blimp-1 in Hodgkin/ReedSternberg cells: a potential pathogenetic lesion in Hodgkin lymphomas. Am. J. Pathol. 173, 242252 (2008).

Acknowledgements

I am grateful to the Deutsche Forschungsgemeinschaft, the Deutsche Krebshilfe, the Wilhelm Sander Stiftung, and the Deutsche Jos Carreras Leukmie-Stiftung for generous support. I thank M.-L. Hansmann and all members of the group for many stimulating discussions. I apologize to all colleagues whose work could not be cited owing to space restrictions.

DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene ATM | BAD | BCL2 | caspase 10 | caspase 8 | FADD | FAS | NFKBIA | NFKBIE | TCF3 | TNFAIP3 | TP53 OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=OMIM Hodgkins lymphoma UniProtKB: http://www.uniprot.org 1-antitrypsin | activation-induced cytidine deaminase | AKT1 | BCL-3 | BCL-6 | BIRC4 | CCL17 | CCL20 | CCL22 | CCL28 | CCL5 | CD4 | CD40L | CFLAR | chromobox protein 8 | colony-stimulating factor 1 receptor | DDR2 | deltex 1 | E12 and E47 | EBF1 | EED | eotaxin | ERK1 | ERK2 | ERK5 | EZH2 | fascin | galectin 1 | GATA2 | GATA3 | granzyme B | IB | ID2 | IKK | IKK | IL-13 | IL-21 | IL-21R | IL-5 | IL-8 | jagged 1 | JAK2 | JUN | JUNB | MSPR | musculin | NEMO | NF-B | notch 1 | PAX5 | PD1 | PDGFRA | POU2AF1 | POU2F2 | PU.1 | REL | RYBP | SOCS1 | STAT3 | STAT5A | STAT5B | STAT6 | TNFRSF11A | TNFSF13 | TNFSF13B | TNFRSF13B | TNFRSF17 | TNFRSF5 | TNFRSF8

FURTHER INFORMATION
R. Kppers homepage: http://www.uni-duisburg-essen.de/ home/fb/ifz/startseite/de_index.shtml
all linkS are aCTive in THe online PdF

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