Symposium article
Annals of Oncology 13 (Supplement 1): 11–18, 2002 DOI: 10.1093/annonc/mdf601
Biology of Hodgkin’s lymphoma R. Küppers1,2*, I. Schwering1,2, A. Bräuninger3, K. Rajewsky1 & M.-L. Hansmann3 1
Institute for Genetics and 2Department of Internal Medicine I, University of Cologne, Cologne; 3Department of Pathology, University of Frankfurt, Frankfurt/ Main, Germany
Significant progress has been made in recent years in our understanding of the cellular origin of Hodgkin and Reed–Sternberg (HRS) cells in Hodgkin’s lymphoma (HL). It is now clear that in most instances HRS cells represent clonal populations of transformed germinal centre (GC) B cells. While the tumour cells in the lymphocyte predominant type of the disease resemble mutating and antigenselected GC B cells, there is evidence that HRS cells in classical HL originate from pre-apoptotic GC B cells. HRS cells of the recently defined novel subtype lymphocyte-rich classical HL moleculary resemble HRS cells of the other types of classical HL, but there appear to be phenotypic differences. In rare cases, HRS cells derive from T cells. In contrast to previous speculations, cell fusion apparently does not play a role in the generation of the tumour clone. By gene expression profiling of HL cell lines, it became evident that HRS cells have lost most of the B cell-typical gene expression program, which may explain why these cells can persist without B cell receptor expression and which suggests that at least one of the transforming events involved in HL pathogenesis affects a master regulator of cell lineage identity. Key words: angioimmunoblastic lymphadenopathy, cell fusion, composite lymphoma, Hodgkin’s lymphoma, Reed–Sternberg cell, somatic hypermutation
Introduction Among haematological malignancies, Hodgkin’s lymphoma (HL) was one of the most enigmatic diseases. Not only the cellular origin of the presumed tumour cells, the mononucleated Hodgkin and the multinucleated Reed–Sternberg cells, but even their clonal nature was unclear for a long time. This was mainly due to the rarity of the Hodgkin and Reed–Sternberg (HRS) cells in the tissue, which hampered the application of many standard molecular biological techniques, and to the unusual (immuno-) phenotype of the HRS cells in classical HL, which did not resemble any normal hematopoietic cell. Only in the lymphocyte predominant (LP) subtype of HL did the HRS cells exhibit a phenotype consistent with an origin from mature B cells [1]. By isolating single HRS cells from biopsy specimens and applying single-cell PCR techniques, considerable progress regarding the biology of HRS cells has been made in recent years. The present article is focused on recent work relating to the nature of HRS cells done in our groups. For a more general overview over the field, the reader is refered to recent reviews
*Correspondence to: Dr R. Küppers, Department of Internal Medicine I, University of Cologne, LFI E4 R706, Joseph-Stelzmann-Str. 9, D-50931 Cologne, Germany. Tel: +49-221-478-4490; Fax: +49-221-478-6383; E-mail:
[email protected]
© 2002 European Society for Medical Oncology
[2, 3] and to the other articles published in this issue of Annals of Oncology.
Origin of HRS cells The origin of HRS cells became clear when techniques were established to isolate single HRS cells from biopsy specimens and analyse these cells for rearranged immunoglobulin (Ig) genes by single-cell PCR [4]. As Ig variable (V) region gene rearrangements are only found in B cells, and as each B cell carries distinct Ig gene rearrangements [5], they represent ideal markers to determine the B cell origin of a cell and the clonal relationship of B lineage cells. Although initial studies reported contradictory results regarding the clonality and B cell origin of HRS cells (reviewed by Küppers and coworkers [6, 7]), it is now well established that HRS cells in classical as well as LPHL represent clonal populations of B cells (see below for a rare T cell origin) [3, 4, 8–12]. Interestingly, HRS cells of both types of HL carry somatic mutations in their V region genes [8–11]. Such mutations occur in the course of immune responses in germinal centres (GC) of lymphoid organs by a process called somatic hypermutation [5, 13]. Somatically mutated Ig genes are hence a hallmark of GC B cells and their descendents, i.e. memory B cells and most plasma cells. In LPHL, intraclonal V gene diversity due to ongoing mutation in the tumour clone was observed in most cases [8, 10, 12], suggesting that these HRS cells represent
12 transformed GC B cells, which is also supported by the immunophenotype of the cells [1]. In classical HL, there was no ongoing mutation, but peculiar features of the mutation patterns suggest a GC B cell origin also for this type of the disease: in about one-quarter of the cases analysed by our group, mutations were found that rendered originally functional Ig gene rearrangements nonfunctional (reviewed by Küppers and coworkers [3, 6]). Such ‘crippling’ mutations included nonsense mutations (i.e. mutations resulting in generation of stop codons) and deletions that resulted in reading-frame shifts. As GC B cells acquiring such mutations are usually removed very efficiently by apoptosis within the GC and not allowed to differentiate further, the respective HRS cells most likely derived from crippled, preapoptotic GC B cells. The presence of obviously crippling mutations in about 25% of cases of classical HL was recently confirmed by another large study (the title of the publication is misleading, as it ignores this result) [11]. As nonsense mutations and deletions represent only a fraction of the unfavourable mutations that result in apoptosis of GC B cells (most cells die because of replacement mutations that impair antigen receptor expression and/or high affinity binding to the respective antigen), we speculate that HRS cells in classical HL as a rule derive from pre-apoptotic GC B cells [6, 9]. Recently, a case of classical HL with unmuated Ig genes was described [14]. Such cases are indeed expected in the scenario of a derivation of HRS cells from the pool of preapoptotic GC B cells: GC founder cells acquire the propensity for apoptosis even before the onset of somatic hypermutation [15], and it is likely that the pool of pre-apoptotic GC B cells includes unmutated GC B cells that are driven into apoptosis because they fail to compete successfully for survival signals with their companions that have already acquired affinityincreasing mutations.
phenotype of HRS cells is different from the phenotype of both the HRS cells of classical HL (BCL6–/+, BOB1–, Oct2–, CD138+) and LPHL (BCL6+, BOB1+, Oct2+, CD138–) (Table 1). By micromanipulation and single-cell PCR for rearranged Ig genes, clonal expansions of HRS cells with mutated Ig gene rearrangements were identified in all cases analysed. Among clonally related rearrangements, no significant intraclonal diversity was observed. Thus, the mutation pattern of rearranged Ig genes reveals similarities to classical HL and differences from LPHL (Table 1). In one case, a population of CD30+ Epstein–Barr virus (EBV)-infected HRS-like cells was observed in addition to the dominant HRS cell clone, suggesting development of an expanded population of EBVinfected (HRS-like) cells in the HL microenvironment, as has also recently been described in other diseases [18, 19].
Identifying common B cell tumour precursors in composite lymphomas In rare cases, HL and B cell non-Hodgkin lymphomas (NHL) occur in the same patient. Such composite lymphomas allow one to study the relationship between HL and B cell NHL. Six combinations of classical HL and B cell NHL have so far been analysed in detail. Whereas in one case, where a B cell NHL occurred several years after a HL, the lymphomas were clonally unrelated to each other [20], in the other five cases, a common B cell progenitor was identified [21–23]. Importantly, in each of these cases, the HRS and B cell NHL clones carried shared as well as distinct somatic mutations. This strongly indicates that the common tumour precursor was a GC B cell, and that the lymphomas developed from two distinct daughter cells of this precursor. Hence, these cases support the idea that HRS cells in classical HL derive from GC B cells and demonstrate the close relationship between HL and B cell NHL.
Origin of HRS cells in lymphocyte-rich classical HL
Rare T cell origin of HRS cells
The term ‘classical HL’ initially included the histological subtypes nodular sclerosis, mixed cellularity and lymphocyte depletion. Recently, the existence of another distinct subtype of classical HL was proposed [16, 17]. This type, lymphocyterich classical (LRC) HL, is characterised by a histology resembling LPHL while the HRS cells display an immunophenotype typical for HRS cells of classical HL (CD30 and CD15 expression and only rare CD20 expression) [16]. We further characterised the immunophenotype of the HRS cells of LRCHL and analysed the rearranged Ig genes of HRS cells of four cases to determine the relationship to classical and LPHL (A. Bräuninger, H.-H. Wacker, K. Rajewsky et al., unpublished data). The HRS cells of all four cases were negative for BCL6 and BOB1 expression. CD138 was only expressed in a minor fraction of HRS cells of one case, while Oct2 expression was detected in two cases. This immuno-
The regular detection of clonal Ig gene rearrangements in HRS cells of classical HL established the B cell origin of these cells in the vast majority of cases. However, as a fraction of HL cases show T cell marker expression (e.g. granzyme B or perforin), we studied three such cases for a potential T cell origin of the tumour cells by analysing micromanipulated HRS cells for T cell receptor (TCR) β and Ig gene rearrangements. Clonal Ig gene rearrangements were detected in two cases and clonal TCRβ gene rearrangements were amplified in one case [24]. Likewise, in another study, two of 13 cases with T cell marker expression showed TCRγ gene rearrangements, while the HRS cells in the 11 other cases were of B cell origin [25]. Thus, in rare instances, HRS cells are derived from T cells. The B cell origin of most cases of HL expressing T cell markers further illustrates the aberrant gene expression profile of HRS cells (see below).
13 Table 1. Distinctive features of the HRS cells of classical and LRCHL and the lymphocytic and histiocytic cells of LPHL Classical HL
LPHL
LRCHL
CD30
+
–
+
CD15
+
–
+
CD20
–/+
+
–/+
BCL6
–/(+)
+
–
CD138
+
–
–/+
Oct2
–
+
+/–
BOB1
–
+
–
EBV infected
∼40% of cases
–
A fraction of cases
Mutated
Yes
Yes
Yes
Intraclonal diversity (variant sequences/all sequences)
Very rarely (4/313)
Frequently (23/81)
Very rarely (2/154)
‘Crippling’ mutations
In ∼25% of cases
Rarelya
Unknownb
Immunophenotype
Rearranged Ig genes
a
One subclone in 16 cases analysed. Number of cases analysed so far too low to determine frequency. Presently one crippled subclone in four cases analysed.
b
HRS cells as cell fusions? Based on the frequent detection of additional chromosomal copies even in the mononucleated Hodgkin cells and the unusual immunophenotype of the HRS cells, which often shows coexpression of markers typical for distinct hematopoietic cell types, it has been speculated that the HRS cell tumour clone may stem from a cell fusion [26–28]. Indeed, the regular detection of clonal Ig (or in rare cases TCR) gene rearrangements does not rule out the possibility that the tumour clone may derive from a fusion between a B (or T) and a non-B cell. To address this issue, we analysed five cases of classical HL of B cell origin for the presence of Ig loci in germline configuration in addition to two rearranged (i.e. B cell derived) Ig loci [29]. This is based on the premise that most B cells carry two rearranged IgH alleles (two VHDHJH or one VHDHJH and one DHJH), which represent clonal markers for distinct alleles, whereas non-B cells have their IgH alleles in germline configuration (Figure 1). However, in none of the five cases were germline IgH alleles observed in addition to two rearranged alleles. Likewise, in a case of T-cell derived HL, no TCRβ alleles in germline configuration were found in addition to two clonal TCRβ gene rearrangements [29]. Thus, these results argue against a role of cell fusion in HRS cell generation.
Searching for transforming events in HRS cells To search for oncogenes or tumour suppressor genes involved in the pathogenesis of classical HL, several candidate genes were analysed for somatic mutations in micromanipulated
HRS cells. Mutations of the tumour suppressor gene p53 appear to be a rare event in HRS cells, as mutations were found in HRS cells in only three of 38 cases analysed in three studies [30–32]. Moreover, as the study by Maggio et al. [31] used pools of HRS cells, it remains unclear whether the three mutated cases described in their work harboured the mutations in all HRS cells or only in a fraction of them. More than 10% of GC B cell-associated NHL show somatic mutations in the CD95 (Fas) gene, which may rescue the lymphoma cells from killing by CD95 ligand-expressing T cells [33]. Interestingly, rare mutations in the CD95 gene can also be found in normal GC B cells (mainly in the 5′ non-coding part of the gene), suggesting that CD95 may, besides bcl-6, represent another non-Ig gene that can be targeted by somatic hypermutation [34]. These findings prompted us to analyse HRS cells from 10 cases of classical HL for mutations in the 5′ region and exon 9 (coding for the signal-transducing death domain) of the CD95 gene. One case harboured three clonal mutations in the 5′ region of the gene, and one other case showed a replacement and a nonsense mutation in exon 9 (defining two subclones of the HRS cell clone) [35]. None of seven cell lines established from HL patients showed Fas gene mutations [35, 36]. Thus, CD95 mutations in the functionally important death domain occur only in rare cases of classical HL, but may in these cases be involved in the pathogenesis of the disease. This is also suggested by the recent observation that patients with germline mutations of the CD95 gene have a 50-fold increased risk of developing HL [37]. HRS cells show constitutive activity of the transcripton factor nuclear factor (NF) κB [38]. As inactivation of this factor in HL cell lines results in apoptosis of the cells [39, 40], NFκB activity may represent an important survival factor for HRS
14
Figure 1. Typing the configuration of the IgH loci in HRS cells to analyse these cells for a potential origin from cell fusion. On the left, the principle of the experiment to test whether HRS cells stem from a cell fusion is depicted. B cells usually carry either two VHDHJH rearrangments or a VHDHJH and a DHJH joint, whereas non-B cells have the IgH locus in an unrearranged configuration. Hence, if the HRS tumour clone originates from a fusion of a B cell with a non-B cell, one would expect to find more than two distinct IgH alleles, i.e. IgH alleles in germline configuration in addition to two rearranged IgH alleles. Please note that this experiment is designed to address the question of whether the HRS tumour clone derives from a cell fusion event, and not to analyse whether Reed–Sternberg cells represent fusions of Hodgkin cells. Regarding this latter issue, there is evidence that Hodgkin cells give rise to Reed–Sternberg cells by endomitosis [63, 64]. On the right, the PCR strategy for the experiment is depicted. HRS cells were analysed for VHDHJH rearrangements using VH and JH primers, for DHJH joints using DH and JH primers and for germline configuration using primers located upstream and within the JH1 gene segment (this fragment around JH1 is indicative of germline configuration as it is deleted in nearly all DHJH and in all VHDHJH rearrangements).
cells. The finding of destructive somatic mutations in the inhibitor of NFκB, IκBα, in two HL cell lines [41–43] prompted the analysis of primary HRS cells. Cabannes et al. [41] detected mutations in two of eight cases, Emmerich et al. [42] found only one mutated case among 10 analysed, and we detected clonal mutations of the IκBα gene in two of five cases (in one case on both alleles) [43], suggesting a role of deleterious IκBα mutations in the pathogenesis of HL in a fraction of cases. As one of the studies was restricted to the detection of large deletions or insertions in the gene [41], and the work by Emmerich et al. was restricted to the analysis of three of the five IκBα exons [42], the frequency of HL cases with IκBα mutations may be underestimated by these analyses.
Crippling Ig V gene mutations and loss of B cell receptor expression in HRS cells HRS cells in classical HL lack detectable Ig gene transcription, and this is probably due to the downregulation of a
number of transcription factors that regulate Ig gene transcription (Oct-2, Bob-1, Pu-1) [11, 44–48]. It has been argued that these findings dismiss the concept that the lack of Ig gene transcription in HRS cells is due to crippling Ig mutations [11, 26– 28, 46]. However, this is a misinterpretation of the scenario that was originally proposed for the cellular origin of HRS cells [6, 9]. The finding of cases of classical HL with crippled Ig receptors was the basis for a speculation about the histogenetic origin of HRS cells unrelated to the question of Ig transcription. ‘Crippled GC B cells’ were defined as cells that had lost the capacity to be positively selected by expression of a high-affinity B cell receptor (BCR), due to various types of unfavourable somatic mutations [6, 9]. As most unfavourable mutations (e.g. replacement mutations resulting in decreased affinity) as such do not abolish Ig transcription, the scenario of a crippled GC B cell origin of HRS cells did not include any assumptions about Ig transcription in these tumour cells [6, 9]. It is nevertheless an interesting question whether the acquisition of crippling mutations and downregulated Ig tran-
15
Figure 2. Generation of crippled B cells in HL and in EBV-positive B cell clones in AILD. This figure compares potential pathways for the generation of crippled B cells in classical HL and among EBV-infected B cells in T cell lymphoma of AILD type. Circles represent B cells, horizontal lines represent V gene rearrangements, vertical lines represent somatic mutations, a small circle represents EBV and an ‘x’ denotes a crippling mutation. In ∼40% of cases of classical HL, HRS cells are EBV infected, suggesting a role of EBV in the survival of crippled B cells in such cases of HL. In classical HL, HRS cells are probably derived from the pool of pre-apoptotic GC B cells, some of which carry obviously crippling mutations like those generating stop codons and others of which carry other unfavourable mutations, such as replacement mutations, which result in reduced affinity to the respective antigen.
scription are related. The molecular features of somatic hypermutation argue against the idea that (crippling) mutations occur after Ig transcription is shut off: somatic hypermutation is closely associated with and dependent on Ig transcription [49, 50], suggesting that it is unlikely that the crippling mutations observed in rearranged Ig genes of HRS cells happened after downregulation of Ig gene transcription. Moreover, if somatic mutations continued to accumulate in HRS cells after loss of Ig transcription, one would expect to find intraclonal sequence diversity in the V genes of the HRS cell clone. This is not observed (see above). Most importantly, even if Ig transcription is first downregulated in HRS cells and then mutations continue to accumulate, these cells would still represent crippled GC B cells, as survival of normal B cells strictly depends on BCR expression [51].
Crippled B cells in T cell lymphoma of AILD type In angioimmunoblastic lymphadenopathy with dysproteinaemia (AILD), one of the most frequent peripheral T cell lymphomas, large numbers of EBV-infected B cells are often found. To determine their clonal composition and differentiation stage, we micromanipulated single EBV-infected cells
from six EBV-rich cases of AILD [18]. Most cells carried mutated Ig gene rearrangements, indicating that EBV preferentially resides in GC and/or memory B cells. In all cases clonal expansions (varying in number and size) among the EBV-infected B cells were observed. Most clonal expansions carried mutated Ig gene rearrangements and ongoing somatic hypermutation was observed in all large clones. Surprisingly, many Ig gene rearrangements in clones with ongoing somatic hypermutation carried crippling mutations [18], indicating that in these EBV-infected B cells somatic hypermutation occurred without any selection for functionality, and even in the absence of the expression, of a BCR. Such an acquisition of somatic mutations has not been described before for B cells in vivo. While this analysis shows that crippled B cells can also develop and persist in diseases other than classical HL, the two settings differ markedly: in HL, the presence of crippling mutations but the lack of intraclonal V gene diversity indicates that the HRS cell precursors represent GC B cells that turned off somatic hypermutation when they underwent malignant transformation (Figure 2). In AILD, it appears that somatic hypermutation was turned on when the EBV-infected B cells started to proliferate, and that this mutational activity continues even after acquisition of destructive mutations. The reason for the aberrant hypermutation activity in the
16 EBV-positive B cells in AILD remains unclear, but may be related to direct or cytokine-mediated cell interactions in the AILD microenvironment, characterised by a dense network of follicular dendritic cells and the CD4+ tumour T cell clone [52].
Loss of the B cell lineage gene expression program in HRS cells One reason for the longstanding controversy about the cellular origin of HRS cells in classical HL was their unusual immunophenotype, which cannot be related to any normal cell of the hematopoietic lineage. On the one hand, typical B lymphocyte markers such as CD19, CD20, CD22 and Ig are either not expressed or only by a small proportion of the malignant cells in a given case [46, 53]. On the other hand, inappropriate lineage marker expression of dendritic cells, monocytes or macrophages (e.g. TARC, CD15, CD83, fascin or restin) can be found in HRS cells [54–57]. An immunophenotype like this is highly unusual for other types of B cell lymphomas. The tumour cells of B cell NHL usually retain the typical B lineage markers and show a differentiation-linked phenotype, as they largely preserve the differentiation state of normal cells upon oncogenic transformation [58, 59]. Gene expression profiles generated by serial analysis of gene expression (SAGE) and microarrays (Affymetrix) from HL cell lines and normal human B cell populations—namely naive B cells, centroblasts, centrocytes and memory B cells— were analysed regarding the B lymphocyte identity of HRS cells (I. Schwering, A. Bräuninger, U. Klein, B. Jungnickel et al., unpublished data). This study revealed a global loss of the B cell lineage gene expression pattern. This concerns genes that are specifically expressed by B cells like Blk and Lyn, two kinases in the BCR signalling pathway, but also genes specific for lymphocytes (e.g. CD52, Pu-B) or hematopoietic cells (e.g. c-src, HPK-1). This feature was consistently observed in three B cell-derived HL cell lines. In addition, an HL cell line of T cell origin was investigated, and downregulation of lymphocyte- and hematopoietic-specific genes was as prominent as in the three B cell-derived cell lines. On the basis of these results we propose a previously unrecognised fundamental defect in maintenance of the differentiation program in HRS cells. Following the hypothesis that the differentiationlinked lymphomagenesis in NHL is a reflection of an intimate linkage between the transforming event(s) and the differentiation program [58, 59], one may speculate that the nature of at least one transforming event in classical HL differs fundamentally from those in B cell NHL. Perhaps, aberrant expression of a master regulator of cell fate decisions in the hematopoietic system could play an important role in this aspect of HL pathogenesis. Intriguingly, expression of Notch 1, which is a transcription factor regulating B cell versus T cell fate decisions in lymphoid precursors by suppressing B cell
development [60, 61], was recently reported to be activated in HRS cells of classical HL [62]. In any case, the global downregulation of the B cell-typical expression profile offers a straightforward explanation for the puzzling finding that the B cell-derived HRS cells may persist without BCR. It is likely that the dependence of BCR expression in a B cell is based on a B cell-specific gene expression program, which is defective or simply lacking in HRS cells. Accordingly, HRS cells might survive without a functional BCR because they have, although originating from a B cell, phenotypically lost their B cell identity.
Acknowledgements We are grateful to Volker Diehl for support and valuable collaborations. Our own work described in this article was supported by the Deutsche Forschungsgemeinschaft through grants SFB502 and BR1238/4.1, and a Heisenberg-Award to R.K.
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