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Hodgkin lymphoma: Pathology and Biology

Stephan Mathas, Sylvia Hartmann, Ralf Küppers

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 Hodgkin lymphoma: Pathology and Biology

Stephan Mathas,a Sylvia Hartmann,b and Ralf Küppersc

a
Max-Delbrück-Center for Molecular Medicine, and Hematology, Oncology, and Tumor

Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany.

b
Dr. Senckenberg Institute of Pathology, University of Frankfurt, Medical School,

Frankfurt/Main, Germany.
c
Institute of Cell Biology (Cancer Research), Medical Faculty, University of Duisburg-Essen,

Essen, Germany.

Address correspondence to Ralf Küppers, PhD, Institute of Cell Biology (Cancer Research),

University of Duisburg-Essen, Medical School, Virchowstr. 173, 45122 Essen, Germany.

Phone: +49-201-7233384; Fax: +49-201-7233386; E-mail: ralf.kueppers@uk-essen.de

Acknowledgement of grant support:

Own work discussed in this review was supported by the Wilhelm Sander-Stiftung, the

Deutsche Forschungsgemeinschaft, the Berliner Krebsgesellschaft, and the Experimental and

Clinical Research Center, a joint cooperation between the Charité Medical Faculty and the

Max-Delbrück-Center for Molcular Medicine, Berlin, Germany.

Conflict of interest: The authors declare no conflict of interest.

1
Abstract

The Hodgkin and Reed-Sternberg (HRS) tumor cells of classical Hodgkin lymphoma (HL), as

well as the lymphocyte predominant (LP) cells of nodular lymphocyte predominant HL

(NLPHL) are derived from mature B cells. However, HRS cells have largely lost their B cell

phenotype and show a very unusual expression of many markers of other hematopoietic cell

lineages, which aids in the differential diagnosis between classical HL (cHL) and NLPHL and

distinguishes cHL from all other hematopoietic malignancies. The bi- or multinucleated Reed-

Sternberg cells most likely derive from the mononuclear Hodgkin cells through a process of

incomplete cytokinesis. HRS cells show a deregulated activation of numerous signaling

pathways, which is partly mediated by cellular interactions in the lymphoma

microenvironment and partly by genetic lesions. In a fraction of cases, Epstein-Barr virus

contributes to the pathogenesis of cHL. Recurrent genetic lesions in HRS cells identified so

far often involve members of the NF- B and JAK/STAT pathways and genes involved in

major histocompatibility complex expression. However, further lead transforming events

likely remain to be identified. We here discuss the current knowledge on HL pathology and

biology.

2
INTRODUCTION

More than 170 years ago, Thomas Hodgkin first described the disease named after him.1 For

several reasons, Hodgkin lymphoma (HL) is, despite recent progress, still one of the most

fascinating hematopoietic malignancies. First, the unique histopathological appearance with

rare lymphoma cells embedded in an inflammatory background represents also today a

technical challenge and impedes rapid advances with respect to its pathogenesis. For a long

time, these technical challenges also prevented the identification of the cellular origin of the

HL tumor cells. Furthermore, the HL phenotype at the crossroads of inflammation and

malignancy is unique among malignancies. Finally, a key driver genomic alteration in HL is

not known. Despite these obstacles, the cellular origin of HL tumor cells has been clarified,

and various molecular and genomic defects were identified which allowed the development of

a concept for HL pathogenesis. In this article, we discuss current concepts on pathology, the

cellular origin, known molecular and genomic defects and the interaction between tumor and

infiltrating surrounding cells in HL.

PATHOLOGY

The main histological feature of HL is the abundance of reactive bystander cells and the

paucity of tumor cells in affected lymph nodes. Four classical HL (cHL) subtypes and nodular

lymphocyte predominant HL (NLPHL) can be distinguished. The malignant Hodgkin and

Reed/Sternberg (HRS) cells in classical subtypes show expression of CD30 (Figure 1a),

MUM1, variable expression of CD15 (Figure 1b) and a typically weak expression of PAX5

(Figure 1c). HRS cells have usually downregulated B cell markers (Figure 1d). In contrast,

tumor cells of NLPHL - the lymphocyte predominant (LP) cells - resemble germinal center

(GC) B cells in their immunophenotype. Whereas the immunophenotype of HRS cells in

classical subtypes is comparable, the microenvironment presents striking differences.

3
Nodular sclerosing HL (NSHL)

This is the most frequent subtype in Western countries (around 80% of cases) mainly

occurring in adolescents.2 NSHL frequently presents with large mediastinal tumors.

Histologically, fibrotic bands confine nodular compartments containing the typical infiltrate

consisting of HRS cells, epithelioid cells, T cells and variable numbers of neutrophils and

eosinophils (Figure 2a). Due to shrinking artifacts the HRS cells can be located in lacunar-like

spaces and are then called lacunar cells (Figure 2b). In NSHL, HRS cells are usually Epstein-

Barr virus (EBV)-negative.

Mixed cellularity HL (MCHL)

This subtype usually occurs in children or elderly people as well as immunocompromised

patients. HRS cells tend to be frequently EBV-infected.2 The affected lymph node presents a

completely effaced architecture with diffuse infiltrates containing histiocytes and eosinophils.

In EBV-negative cases, the histiocytes rather present as epithelioid cells (Figure 2c), whereas

in immunocompromised patients they rather appear as spindle shaped macrophages (Figure

2d).3,4

Lymphocyte depleted HL (LDHL)

This subtype has become exceedingly rare, since with better reagents for PAX5 staining,

PAX5-positive LDHL can now be better distinguished from PAX5-negative (and ALK-

negative) anaplastic large cell lymphoma. In LDHL, HRS cells can be relatively abundant

(reticular variant). Other cases of LDHL represent a diffuse fibrosis in which scattered HRS

cells are embedded. LDHL is more likely to be encountered in immunocompromised patients

(human immunodeficiency virus (HIV)-positive), and HRS cells are frequently EBV-infected.

4
Lymphocyte rich cHL (LRCHL)

This is another rare subtype of HL which usually occurs in adults and shows a prevalence for

cervical lymph nodes and the Waldeyer lymphatic tissue. LRCHL frequently presents with

early stages.5 HRS cells are EBV-infected in 30-50% of the cases.5,6 Histology shows a rather

monomorphic picture with small and often confluent nodules of mantle zone B cells,

frequently containing GCs. HRS cells are embedded in these nodules (Figure 2e). A helpful

diagnostic feature is the presence of rosetting PD1-positive T cells.6 In LRCHL, HRS cells

more frequently express B cell transcription factors (TFs) and CD20 than HRS cells in the

other cHL subtypes.5,6

Nodular lymphocyte predominant HL (NLPHL)

NLPHL most closely resembles LRCHL. However, there are several striking differences in

clinical presentation and morphology. NLPHL shows a strong predominance of the male

gender and more frequent relapses than LRCHL.5,7 The most important difference to LRCHL

are the LP tumor cells, which have a GC B cell phenotype and are usually negative for CD30

and CD15. Morphologically, typical and variant patterns can be distinguished.8 In the typical

pattern, LP cells are located in large nodules of naive B cells and are surrounded by follicular

T helper (TFH) cells (Figure 1e and 2f).9,10 In contrast, the follicular microenvironment is

usually lost in the diffuse NLPHL variants, which show considerable morphologic and

molecular overlap with T cell/histiocyte rich large B cell lymphoma.9,11

Differential diagnoses

Whereas NSHL in young patients usually represents a clear cut diagnosis, MCHL and

LRCHL can be mimicked by T cell lymphomas, particularly in adult or elderly patients.

5
When HRS cells are PAX5-negative, a CD30-positive T cell lymphoma has to be excluded.

Notably, CD30-positive cutaneous lymphoproliferative diseases can closely resemble PAX5-

negative HL when involving lymph nodes.12 In most cases this differential diagnosis can be

solved when the respective clinical information is available, expression of cytotoxic

molecules or HRS-related markers like CD83 (Figure 1f), CCL22, TUBB2B or STAT3 or a

clonal B cell receptor (BCR) or T cell receptor rearrangement can be demonstrated.13,14

However, in some cases this differential diagnosis remains challenging.

Even when HRS cells are PAX5-positive, T cell lymphomas remain the main

differential diagnoses in adult and elderly HL patients. Both in angioimmunoblastic T cell

lymphomas and in peripheral T cell lymphomas of the NOS (not other specified) type the

occurrence of HRS-like cells has been described.15,16 Therefore, particular caution is

necessary, when HRS-like cells are EBV-infected and express B cell markers.

Concerning NLPHL, the main differential diagnosis is a progressive transformation of

GCs. Rosetting of TFH cells is the feature that helps most in distuingishing NLPHL from

progressively transformed GCs.17

CELLULAR ORIGIN

As HRS cells show a very unusual immunophenotype with frequent coexpression of markers

of distinct cell types of the immune system,18 their cellular origin was enigmatic for a long

time. The detection of rearranged immunoglobulin (Ig) genes in isolated HRS cells finally

revealed their B cell origin in the vast majority of cases.19,20 This is because Ig heavy and light

chain gene rearrangements are highly specific for B cells and thus represent a genetic marker

for their origin. The finding of the same rearrangements in all HRS cells of a given case also

demonstrated monoclonality of HRS cells and thus settled a controversial discussion about

their clonality.19,20 Importantly, IgV genes of HRS cells in nearly all cases carried a high load

6
of somatic mutations, and in about 25% of cases, destructive mutations rendered originally

functional rearrangements non-functional.19,21 Somatic mutations are introduced into IgV

region genes of antigen-activated B cells proliferating in GC.22 Destructive mutations

physiologically occur in mutating GC B cells, however, normally their occurrence rapidly

causes apoptotic death of the B cells. Thus, HRS cells frequently or as a rule derive from GC

B cells that acquired disadvantagous mutations and normally would have undergone

apoptosis. Further support for a B cell derivation of HRS cells is that these cells frequently

have undergone class-switch recombination,23,24 a further B cell-specific process, and that in

composite lymphomas of a cHL and a mature B cell non-Hodgkin lymphoma (NHL) these

lymphomas are frequently clonally related and hence stem from the same mature B cell.25

There are also a few lymphomas diagnosed as cHL that carry T cell receptor gene

rearrangements and hence derive from T cells.21 This may indicate that a very small subset of

cases of cHL has a T cell origin, or that T cell lymphomas exist that are histopathologically

and immunophenotypically indistinguishable from cHL. This holds particularly true for

cutaneous T cell lymphomas which can frequently drain to local lymph nodes and evoke

pictures closely resembling cHL.

The GC B cell phenotype of LP cells in NLPHL already indicated their B cell origin.18

This was validated by IgV gene studies of isolated LP cells. The rearrangements lack

destructive mutations and in a fraction of cases show intraclonal diversity of V gene

rearrangements, which indicates low level active somatic hypermutation during clonal

expansion.26,27 These features, together with the expression of typical markers of GC B cells,

the follicular growth pattern, and the global gene expression pattern of LP cells, strongly

support a derivation of LP cells from positively selected GC B cells.18,28

7
GENERATION OF REED-STERNBERG CELLS, HRS PRECURSOR CELLS, AND

HRS CELLS IN THE PERIPHERAL BLOOD

A hallmark of cHL is the presence of mononuclear Hodgkin and bi- or multinuclear Reed-

Sternberg cells. The relationship between these two forms of tumor cells has been unclear for

a long time. Recent time-lapse microscopy studies of HL cell lines showed that Hodgkin cells

frequently undergo incomplete cytokinesis and that Reed-Sternberg cells are generated by re-

fusion of two sister cells that are still connected by microtubuli bonds.29,30 Thus, it appears

that a defect to complete an advanced cytokinesis is the main reason for the generation of

Reed-Sternberg cells from Hodgkin cells. These experiments as well as earlier studies also

revealed that Reed-Sternberg cells have little proliferative capacity, and that Hodgkin cells are

the main proliferative compartment of the HRS tumor clone.29-31 However, perhaps not all

Hodgkin cells have the same proliferative capacity. Based on studies with HL cell lines, there

is indication that a small subset of Hodgkin cells, so-called side population cells that express

ABC transporters and can therefore expell Hochst dye 33342, have features of tumor stem

cells and can reestablish the HRS cell clone in subcloning experiments.32,33 However, as side

population cells were not seen in all HL cell lines, the general role of these cells for HL

remains unclear.

Another interesting issue is whether the tumor clone in HL also encompasses members

that are not identifiable as CD30+ cells with the typical morphology and phenotype of HRS

cells, and whether HRS cell clone members can be found in the peripheral blood. In an

analysis of two HL patients with advanced disease, IgV gene rearrangements of the HRS cells

were detected in multiple tissues, but even with a highly sensitive approach not in the

peripheral blood.34 Another study reported about the presence of CD20- and BCR-positive,

but CD30-negative HRS clone members in the peripheral blood,35 but the validity of that

study was critizised based on technical concerns.36 In a further recent study, IgV gene

8
rearrangements thought to stem from the HRS cells were detected in a few of the tested

samples in peripheral blood mononuclear cells, but much more frequently in serum.37

Because of the presence of HRS cell DNA in the serum, it needs to be clarified whether the

rearrangements detected in the cell fraction are really derived from circulating HRS cells, or

from contaminating HRS cell DNA in the serum. Further indication for the presence of HRS

cell-derived DNA in circulating cell-free DNA stems from a deep sequence analysis for

genomic imbalances detected in the HRS cells in the lymph node tissue.38 Thus, it appears

that DNA from HRS cells can frequently be detected in cell-free blood serum. It is

worthwhile to study whether this can be used for minimal residual disease detection and serve

as a prognostic biomarker.

DEREGULATED TRANSCRIPTION FACTOR NETWORKS AND SIGNALING

PATHWAYS

HRS cells are characterized by a complex network of deregulated TFs, which interfere with

cellular differentiation, reflect their activated phenotype, and coordinate their growth and

survival. The loss of B cell phenotype is mediated by epigenetic silencing of key regulators of

B cell differentiation21,39 in combination with an up-regulation of transcriptional antagonists,

as demonstrated for the E2A blockade by ABF-1 and ID2, or Pax5 inhibition by NOTCH1.40-
42
Whereas one group of B-cell TFs (e.g. OCT2, PU.1, BOB.1, FOXO1, ETS)43-46 is nearly

completely lost in an unifying manner, the expression of others is maintained (PAX5),47 in

some instances even at high-level expression (e.g. E2A, IRF4).41,48 Although genetic

alterations for some TFs were described,46,49 a rather functional disruption of B cell-specific

gene expression as lead defect in HRS cells is further corroborated by i) maintenance of B

cell/plasma cell gene expression (e.g. CD20, BCL6, BLIMP1) in a subfraction of HL cases, 50-
52
and ii) reactivation of B cell genes following e.g. FOXO1, EBF1 or CD99

9
reconstitution49,53,54 or NOTCH1 inhibition.40 Furthermore, reconstitution of KLF4 down-

regulated the E2A antagonist ABF-1.55 The role of altered epigenetic regulators such as PcG

family members or lost CBFA2T3 expression for silencing of lineage-specific genes is

unclear.56,57 Finally, transient hypoxic conditions in early stages of cHL pathogenesis might

also contribute to the peculiar phenotype of HRS cells.58

HL was among the first malignancies for which an oncogenic role of NF- B was

recognized. Initially referred to classical NF- B-p65 (RELA),59 it became evident that

combinatorial activities of various NF- B components, including REL, RELB and p52,60-63

mediate its key functions in HRS cells, which reflects the concomitant activation of canonical

and non-canonical NF- B signaling (Figure 3). Presumably facilitated by high-level

expression of various TNF/death-receptor family members such as CD30, CD40 or RANK,

constitutive I B kinase (IKK) complex and NF- B inducing kinase (NIK) activation

contributes to NF- B activity in HRS cells and argues for ongoing upstream signaling.21,60

The JAK/STAT signaling pathway represents a second key pathway in HL. STAT3, STAT5

and STAT6 are activated and (for some factors) expressed at high level in HL (Figure 3).64,65

Disruption of JAK/STAT signaling by genetic interference, inhibition of outside-in signaling

or pharmacological intervention led to growth inhibition and cell death. The high frequency of

genomic alterations as observed for the NF- B pathway (see below) and induction of HRS-

like cells by STAT5 suggest the JAK/STAT pathway as further key driver in HL

pathogenesis.65-68

Third, deregulation of the AP-1/CREB complex constitutes a hallmark of HRS cells.

JUN, JUNB and ATF3 are highly upregulated in HRS cells, contribute to growth and

survival, and regulate HL-specific genes (Figure 3).69,70 At least in the context of the

microenvironment, MAP kinases contribute to their activation.71 Furthermore, IRF TFs are

centrally involved in HL pathogenesis.48,72 IRF4 and IRF5 are consistently expressed in HRS

10
cells and drive their growth and survival; IRF5 orchestrates, together with NF- B, the HL-

specific gene expression pattern.73 There is increasing evidence for an extensive cross-talk

between these TFs. For example, NF- B maintains high-level expression of STAT5, JUNB

and GATA3, and IRF5 transcriptionally upregulates various AP-1/CREB factors.64,73,74

Finally, HRS cells express a unique pattern of TFs asssociated with hematopoietic stem or

precursor cells of other lineages such as GATA2 or HOXB9,75,76 which might be linked to

their unusual phenotype. Given the plethora of TF alterations, unbiased experimental

approaches are required to judge about the importance of individual TF activites and to

further dissect their hierarchical organization, in order to identify the hub TFs required for HL

pathogenesis and key signaling pathways for targeted therapeutic intervention.

Much less is known about signaling pathways and TF alterations in NLPHL. NLPHL

shares signaling pathways with cHL, as demonstrated for NF- B, STAT6 or JAK2 activation,

but is also clearly distinct from cHL.28 For example, AP-1 activation and target gene

expression is absent in most cases,70,77 and the loss of B cell phenotype is much less

pronounced.28,45,52

GENETIC LESIONS

A number of oncogene and tumor suppressor genes were studied in HRS cells for mutations.

These studies revealed a frequent involvement of components of the NF- B signaling

pathway (Table 1). HRS cells recurrently harbor genomic gains of the NF- B factor REL,78,79

and of MAP3K14 (NIK),80,81 a major positive regulator of the alternative NF- B pathway.

The NF- B inhibitors NFKBIA, NFKBIE, and TNFAIP3 are frequently inactivated by point

mutations or deletions.82-87 Additional components of this pathway, including BCL3, CYLD

and TRAF3, were also found to be mutated at low frequency.21 Another signaling pathway

with frequent genetic lesions in several of its components is the JAK/STAT pathway. The

11
JAK2 gene is frequently affected by genomic gains, and rarely by translocations.81,88-90 SOCS1

and PTPN1, encoding main negative regulators of JAK/STAT signaling, are recurrently

inactivated by mutations (Table 1).91,92

Another group of genetic lesions in HRS cells affects MHC expression and interaction

with T cells. The gene of the MHC class II transactivator (CIITA) is recurrently involved in

chromosomal translocations, which appear to inactivate the gene, causing reduced MHC class

II expression.93 The gene for 2-microglobulin (B2M), a component of the MHC class I

complex, carries inactivating mutations in a fraction of cases.94 In line with this, HRS cells

frequently lack detectable MHC class I expression. Furthermore, the chromosomal gains on

9p24.1 affecting the JAK2 gene also involve the PDL1 and PDL2 genes (and in addition

JMJD2C, encoding an epigenetic regulator).66,95 These ligands inhibit cytotoxic T cells when

binding to PD1, which is expressed by activated T cells. Interestingly, in contrast to its unique

morphological appearance, no genomic alteration unique to HL has been reported so far and a

lead genetic alteration, characteristic for many other hematologic malignancies, is still

lacking.

Only little is known about genetic lesions in the LP cells of NLPHL. Translocations of

the proto-oncogene BCL6 are detectable in about 30% of cases, and SOCS1 is mutated in

about half of the cases.18 Similar to other GC-derived B cell lymphomas and cHL, LP cells

frequently show gains of 2p16 (REL locus).11 A recent study revealed three further highly

recurrent genetic lesions in LP cells, each present in about 50% of NLPHL, namely mutations

in the genes for the phosphatase DUSP2, the serine/threonine kinase SGK1, and in JUNB.96

The high number of stop mutations in JUNB correlates well with the absence of AP-1

activation in LP cells of a fraction of NLPHL.70,96

ROLE OF EBV IN HL PATHOGENESIS

12
In about 30-40% of cases of cHL in Europe and North America, the HRS cells are latently

infected by EBV, a herpes virus.97 The infection rate is even higher in childhood cases in

Central and Southern America, where about 80% of cases show an EBV infection of HRS

cells. In EBV-positive cases, usually all HRS cells are positive, indicating that the infection

was an early event in lymphoma development. The EBV+ HRS cells typically show an EBV

latency II gene expression profile, meaning expression of the viral proteins EBV nuclear

antigen 1 (EBNA1) and latent membrane proteins 1 and 2a (LMP1 and LMP2a).97 EBNA1 is

essential for replication of the episomal viral genome in proliferating cells. LMP1 mimics an

active CD40 receptor and hence stimulates NF- B and PI3K/AKT activity.98 LMP2a has a

cytoplasmic motif that resembles the ITAM motif of the BCR. It can therefore mimic an

active BCR.99 As BCR and CD40 signalling are main survival signals for GC B cells, EBV

infection of GC B cells may be a way how GC B cells with destructive mutations survive and

become HRS precursor cells.100 Indeed, in vitro, EBV can rescue crippled GC B cell from

apoptosis,99,101 and all cHL cases with destructive mutations that prevent BCR expression

were found to be EBV-positive.100 Furthermore, EBV-caused infectious mononucleosis has

been reported to be associated with an increased risk of HL.102 These findings indicate a

major pathogenetic role of EBV in the virus-positive cases.

MICROENVIRONMENTAL INTERACTIONS

HRS cells are embedded in an inflammatory microenvironment which consists of various

hematopoietic cell types, including different types of T cells, macrophages, B cells,

eosinophils and others. Due to space limitations, here we can only focus on some aspects of

interactions between HRS and surrounding cells. It is assumed that i) cells of the

microenvironment take part in complex interactions with HRS cells and support their growth

and survival, and ii) the other way around, HRS cells actively influence and modify the

13
composition of the surrounding infiltrate, although experimental evidence for these

assumptions is rather weak and most data are correlative. Interactions might be direct, for

example by CD30, CD40, RANK or CD95 interactions, which contribute to e.g. NF- B

activation in HRS cells and to their immune escape.21 A plethora of interactions act indirectly,

for example the attraction and polarization of T cells by secretion of RANTES, TARC,

CCL22 or galectin-1, or of eosinophils by secretion of RANTES, IL-5 or CCL11.103

Furthermore, also fibroblasts have most likely impact on the microenvironmental composition

and establishment of the respective HL-subtype and contribute to the attraction of infiltrating

cells like eosinophils by production of eotaxin.104

Apart from effects of bystander cells on HRS cell growth and survival, the lymphoma

cells obviously inhibit an effective immune reponse in a complementary manner. This is

highlighted by expression or secretion of PD-1 ligand or galectin-1 which directly interfere

with the functional activity of T cells, or a shift of T cell polarization towards a regulatory

phenotype.95,105,106 It is assumed that the impressive clinical results of patients treated with

PD-1/PDL1 interfering antibodies are based on the reconstitution of an effective T cell

immune response against HRS cells by reversion of the inhibited T cell effector functions. 105

However, serial biopsies in the course of PD-1/PDL1 antibody treatment are still lacking, and

redirection of T cells towards HRS cells following such treatment has not formally been

demonstrated. It could also be speculated that clinical responses following such immune-

checkpoint interference are mainly due to „simple“ disruption of the growth- and survival-

promoting interaction between HRS and surrounding T cells. HRS cells secrete substances,

such as IL-13 or galectin-1, which primarily act on and polarize specific T cell subsets or

actively prevent an effective Th1-response.106,107 Furthermore, an effective T cell-response

might be prevented by MHC defects of HRS cells.93,94

14
 Important lessons can be learned from HL in patients suffering from HIV infection.

Before the era of antiretroviral therapy (ART), when CD4+ T cell counts were usually very

low, a strong increase of aggressive NHL, but not HL, was documented in HIV-patients.108

This changed with the implementation of ART and an increase of CD4+ T cell numbers and

thus an improved immunity. Whereas the incidence of aggressive NHL dropped, the

incidence of HL increased.109 This might be the best in vivo evidence for the importance of

the HRS – T cell interaction in HL. Similarly, despite the high rate of EBV-reactivation and

EBV-associated lymphomas in post-transplant patients, HL is only rarely observed in this

setting.110

Apart from T cells, infiltrating macrophages might support the growth of HRS cells

and prevent an effective immune response towards HRS cells.3,103 HRS cells express at high

level cytokines and chemokines which act on macrophages, and the density of macrophage-

infiltration correlates with prognosis of HL patients.103,111 Together, although we still do not

understand the complexity of interactions between HRS cells and their microenvironment and

their functional role during transformation, the impressive clinical results following disruption

of such interactions indicate its importance for both, to reveal pathogenic mechanisms and to

develop new treatment strategies for HL patients.

ACKNOWLEDGEMENTS

We thank Martin-Leo Hansmann for support and many stimulating discussions. We apologize

to all those colleagues whose work we could not cite due to length restrictions.

15
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29
Figure legends

Figure 1. Typical immunohistochemical features of HL.

a. Expression of CD30 in HRS cells (arrows, membrane bound and golgi field, NSHL,

CD30-immunostaining, 400x).

b. Strong cytoplasmic CD15 expression in HRS cells (arrows, NSHL, CD15-

immunostaining, 400x).

c. Typical weak PAX5 expression in a HRS cell in LRCHL (arrow). Surrounding mantle

zone B cells show a more prominent PAX5 expression (arrow heads, PAX5-

immunostaining, 400x)

d. Weak CD20 expression in an HRS cell, as it is observed in some cases (arrow). The few B

cells in the reactive infiltrate present a strong CD20 expression (arrow heads, CD20-

immunostaining, 400x).

e. PD1-positive T cell rosettes surrounding LP cells in NLPHL (arrows, PD1-

immunostaining, 200x)

f. CD83 expression in HRS cells (arrow, MCHL, CD83-immunostaining, 400x)

Figure 2. Morphologic characteristics of different HL subtypes

a. Typical nodule of NSHL confined by a sclerotic band (arrow heads) and containing HRS

and lacunar cells (arrows, hemalum-eosin staining (HE), 100x)

b. HRS cells in NSHL – so called lacunar cells (arrows, HE, 200x).

c. MCHL with a typical mixed reactive infiltrate consisting of epithelioid cells, lymphocytes

and eosinophils (HE, 400x). HRS cells are marked with arrows.

d. MCHL in an immunocompromised patient with abundant spindle cell shaped histiocytes

(arrow heads, HE, 200x) HRS cells are marked with arrows.

30
e. HRS cells surrounded by small B cells in LRCHL (HE, 400x). HRS cells are marked with

arrows.

f. LP cells (arrows) in a mixed inflammatory infiltrate containing B cells, TFH cells and

epithelioid cells (HE, 400x).

Figure 3. Key signaling pathways in cHL

Scheme of the main signaling pathways involved in cHL pathogenesis. In part by activation

of various cell surface receptors, as indicated, or the EBV protein LMP1, the canonical and

non-canonical NF- B pathways are activated, resulting in nuclear translocation and

transcriptional activity of various NF- B members including p50/p65, p52, RELB or BCL3.

A second major pathway is the JAK/STAT signaling pathway, as documented for high-level

STAT3, STAT5 and STAT6 activation in HRS cells in at least a fraction of cHL cases.

Further key players in the TF landscape of HRS cells are IRF4 and IRF5, the activation

mechanism and composite binding site partners (marked by „X“) of which are not yet

clarified and which coordinates the HL-specific gene expression program, high-level

activation of various AP-1/CREB members (JUN, JUNB, ATF3) and NOTCH1. Known

interactions, i.e. synergistic regulation of target genes or subordinate activation of respective

TFs, are indicated by green arrows. Known genomic lesions of HRS cells include various

negative regulators of these signaling pathways such as A20, I

Bold circles indicate signaling components directly involved in transcriptional regulation.

31
Table 1. Recurrent genetic lesions in HRS cells

Gene Types of lesions Affected cases/ Remark References

cases analyzed

(%)

NF- B pathway
82,84,112
NFKBIA SNVs, deletions 5/23 (22)
83
NFKBIE SNVs, deletions 1/6

TNFAIP3 SNVs, deletions 35/79 (44) Frequency in EBV- cases 85-87

about 70%
78,79
REL gains 33/71 (46)
80,81
MAP3K14 gains 18/69 (26)

JAK/STAT pathway
92
SOCS1 SNVs, deletions 8/19 (42)
91
PTPN1 SNVs, deletions 6/30 (20)
81,88,90
JAK2 gains 30/77 (39) JAK2 gains also involve the

neighboring genes PDL1,

PDL2, and JMJD2C

Antigen presentation
93
CIITA translocations 8/55 (14)
94
B2M SNVs, deletions 7/10 (70)
113
CD58 deletions 3/13 (23)*

Others
18
TP53 SNVs, deletions 3/39 (8)
18
FAS SNVs, deletions 2/30 (7)

32
 114
MDM2 gains 4/6 (67)

SNVs, single nucleotide variants; *plus inactivating mutations in three of seven HL cell lines

33
34
Figure 3

35