Copyright Blackwell Munksgaard 2005

Eur J Haematol 2005: 75 (Suppl. 66): 2633

All rights reserved

EUROPEAN
JOURNAL OF HAEMATOLOGY

Pathogenesis of Hodgkins lymphoma

Kuppers R, Schmitz R, Distler V, Renne C, Brauninger A, Hansmann M-L.
Pathogenesis of Hodgkins lymphoma.
Eur J Haematol 2005: 75 (Suppl. 66): 2633. Blackwell Munksgaard 2005.
Abstract: In Hodgkins lymphoma (HL), the B cell origin of the tumour
cells, the Hodgkin and Reed-Sternberg (HRS) cells, has been disclosed by
molecular single cell analysis about 10 yr ago. This nding formed the
basis for various studies aimed to better understand the pathogenesis of
this peculiar malignancy and the pathophysiology of the HRS cells.
Work of our groups in this regard was focussed recently on two main
topics, namely the study of dierential gene expression in HRS cells and
the pathogenesis of composite lymphomas. Composite lymphomas are
combinations of HL and B cell non-Hodgkin lymphomas, that turned
out to be often clonally related. By molecular analysis of several composite lymphomas for potential transforming events, we identied
examples of both shared as well as distinct transforming events. Comparing gene expression proles of HL-derived cell lines with the corresponding proles from other B cell lymphomas and normal B cell subsets
revealed a global down-regulation of the B cell-specic gene expression
signature in HRS cells. Moreover, we identifed aberrant expression and
activity of multiple receptor tyrosine kinases in HRS cells of classical and
to a lesser extend lymphocyte predominant HL, which appears to be a
unique feature of HL, and may oer novel strategies for treatment.

In Hodgkins lymphoma (HL), the Hodgkin and

Reed-Sternberg (HRS) cells, which represent the
tumour cells of this malignancy, usually account for
less than 1% of cells in the tumour tissue. This has
for a long time hampered their molecular analysis.
However, through the establishment of microdissection techniques to isolate HRS cells and the
analysis of single HRS cells by sensitive PCR
methods, it became possible to study these cells at
the molecular level. These studies revealed the
clonality and B cell nature of the HRS cells and
provided evidence that HRS cells are derived in
most cases from a subset of germinal centre (GC) B
cells (13). Only in rare cases, HRS cells derive
from T cells (4, 5). Microdissection and single cell
PCR methods were also instrumental to study HRS
cells for potential transforming events, such as
mutations in proto-oncogenes or tumour suppressor genes (68). Recently, we applied these methods
to the analysis of composite lymphomas, which are
rare combinations of a HL and a B cell nonHodgkin lymphoma (NHL). The results of this
analysis will be discussed below.
Due to the problems in the analysis of primary
HRS cells, extensive eorts were undertaken to
establish cell lines from HRS cells as in vitro models
26

Ralf K
ppers1, Roland Schmitz1,

Verena Distler1,2, Christoph Renn2,
Andreas Bruninger2, Martin-Leo
Hansmann2
1
Institute for Cell Biology (Tumour Research), University
of Duisburg-Essen, Medical School, Essen, Germany;
2
Department of Pathology, University of Frankfurt,
Frankfurt, Germany

Key words: Hodgkin's lymphoma; composite lymphoma;

receptor tyrosine kinase; Reed-Sternberg cell, Epstein
Barr virus, p53, bcl-1, bcl-2
Correspondence: Ralf K
ppers, Institute for Cell Biology
(Tumour Research), University of Duisburg-Essen,
Medical School, Virchowstr. 173, 45122 Essen, Germany
Tel: +49-201-7233384
Fax: +49-201-7233386
e-mail: ralf.kuppers@uni-essen.de

of these cells. This turned out to be extremely

dicult, and in the last 30 yr, only about 10 cell
lines were established that are still existing. Since
the attempts to establish cell lines were so rarely
successful, it is an important question whether the
lines are derived from the HRS cells in the patient,
and whether the few lines are indeed representative
of HRS cells (notably, all cell lines were derived
from blood, bone marrow or pleural eusion, but
none from lymph node biopsies). Indeed, only for
one of the lines, L1236, the derivation of the cell
line from the HRS cells in the patient was proven
(9). Nevertheless, several important new ndings
about the pathogenesis of HL were initially identied analysing HL cell lines and were then
conrmed for primary HRS cells. For example,
we performed gene expression proling with several
HL cell lines, and the main ndings of that study
were conrmed for HRS cells in lymph node
biopsies, as discussed below (1012).
Pathogenesis of composite lymphomas

In rare cases a combination of classical HL and B

cell NHL occurs in the same patient, either
concurrently or sequentially. Important insights

Pathogenesis of Hodgkins lymphoma

into the development of those composite lymphomas were provided by single cell PCR analysis for
rearrranged Ig V genes of the two lymphomas.
Thus, clonal relationship between the two dierent
tumours was demonstrated in the majority of the
cases analysed (1320). Interestingly, in many of
the clonally related cases, the rearranged V genes of
the two lymphomas showed both shared as well as
distinct somatic mutations in the HRS and the
NHL cells. This strongly indicates that in these
cases the two lymphomas developed separately
from a common (premalignant) precursor and that
these cases do not represent a transformation of
one of the lymphomas into the other. Moreover,
since somatic mutations are introduced into rearranged V genes of B cells participating in germinal
centre (GC) reactions (21), the V gene mutation
pattern in these cases suggests that decisive steps in
the malignant transformation process happened in
distinct members of a proliferating GC B cell clone.
The molecular analysis of such clonally related
composite lymphomas permits the unique opportunity to study the time point and the role of
transforming events in the multistep process of
lymphomagenesis, to gain insights into the development of two distinct malignancies from a common
precursor and to better understand the pathogenesis
of HL. Six clonally related composite lymphomas
were analysed for transforming events that have
been described to be present in cases of HL and/or BNHL (manuscript in preparation) (Table 1).
Hallmarks of many B-NHL are chromosomal
translocations, juxtaposing one of the immunoglobulin loci with one of several proto-oncogenes
(22). However, in HL, Ig-associated chromosomal
translocations have not been determined on a

molecular level so far. Interestingly, in three cases

of composite lymphomas combining HL with
mantle cell lymphoma (MCL) and HL with follicular lymphoma (FL) identical hallmark translocations (IgH/bcl-1 and IgH/bcl-2) were obtained from
the B-NHL and the corresponding HL (Table 1).
Further sequence analysis of the bcl-2 and bcl-1
translocational breakpoints showed loss of nucleotides and addition of non-germline encoded nucleotides (typical for VDJ recombination) directly at
the junctions of IgH and the translocated oncogenes in both NHL and HL. Hence, the common B
cell precursor of the composite lymphomas most
likely acquired these Ig-associated translocations
during misguided VDJ recombination in an early
developmental step.
Mutations in the tumour suppressor genes IjBa
CD95 and ATM have been found in a fraction of
cases of HL or NHL and presumably contribute to
the pathogenesis of these malignancies (6, 2327).
However, in none of the composite lymphoma
cases analysed mutations in these genes were
detected in the HL or the NHL.
A remarkable nding of the analysis of transforming events was the restriction of two clonal
replacement mutations in the p53 gene to the
DLBCL cells of a composite lymphoma case
combining HL and DLBCL (Table 1). Those two
mutations were demonstrated to be located on
dierent alleles and were found at positions that
have been previously reported to be frequently
mutated in aggressive NHL, suggesting a pathogenic role for these mutations (28, 29). The presence
of the mutations exclusively in the DLBCL suggests
a late transforming event, which happened most
likely in the GC because of the occurrence of shared

Table 1. Characteristics of six clonally related composite lymphomas1

Case
1
2
3
4
5
6

Composite
lymphoma

HL and NHL present

in the same tissue

Mutated
V genes

Shared and distinct

V gene diversity

Intraclonal
V gene diversity

EBV

IgH-associated
translocations2

P53 gene
mutations

HL
FL
HL
FL
HL
B-CLL
HL
SMZL
HL
MCL
HL
DLBCL

Yes

Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
Yes
Yes

Yes

No
Yes
No
Yes
No
No
No
No
Two subclones
No
No
No4

No
No
No
No
No
No
No
No
Yes (one subclone)
No
No
No

Igh/bcl-2
Igh/bcl-2
Igh/bcl-2
Igh/bcl-2

Igh/bcl-1
Igh/bcl-1

No
No

No
No
No
Two clonal mutations
on separate alleles

Yes
Yes3
No3
Yes3
Yes

Yes
Yes
No
Only distinct
Yes

The data on the rearranged Ig V genes and the EBV status are taken from Refs 14, 15, 1719.
For each of the lymphomas, the breakpoint in the HRS cells and B-NHL cells was identical.
3
NHLs were first diagnosed 315 yr before diagnosis of the composite lymphomas.
4
Indication of two copies of the same clonal rearrangement with distinct mutation patterns was obtained in the previous single cell analysis (from Ref. 17).
: not analysed; HL: Hodgkin's lymphoma; FL: follicular lymphoma; B-CLL: B cell chronic lymphocytic leukemia; SMZL: splenic marginal zone lymphoma; MCL: mantle cell
lymphoma; DLBCL: diffuse large cell lymphoma.
2

27

Kuppers et al.
and distinct somatic V gene mutations in HRS as
well as DLBCL cells. This late separate transforming event might have prompted the evolution of the
DLBCL from a common precursor in terms of
separation of the two lymphoma clones.
In conclusion, the molecular analysis of several
composite lymphomas supports the concept that the
development of such lymphomas is characterised by
both early shared transforming events and distinct
events occurring at later stages that then determine
the development of the dierent malignancies.
The role of EpsteinBarr virus in HL pathogenesis

EpsteinBarr virus (EBV), a member of the c-herpes

virus family, infects more than 90% of the human
population worldwide. The reservoir of the virus are
B cells, in which a latent infection is established. The
EBV normaly is a harmless passenger, but in rare
cases, EBV is also involved in the pathogenesis of
malignancies, mainly B cell lymphomas. One of
these is HL, as EBV is found in the HRS cells in
about 40% of cases of classical HL in the Western
world (30). In EBV-harbouring HRS cells, three
EBV-encoded latent proteins are expressed, namely
the EBV nuclear antigen 1 (EBNA1) and the latent
membrane proteins 1 and 2a (LMP1 and LMP2a)
(30). The EBNA1 is required for the replication of
the circular viral genome in proliferating cells. The
LMP1 mimics an activated CD40 receptor, which
plays a major role in the interaction of B and T cells
and the survival of GC B cells. Indeed, LMP1 is a
viral oncogene, because LMP1 transgenic mice
develop B cell lymphomas (31). The cytoplasmic
domain of LMP2a contains an ITAM (immunoreceptor tyrosine-based activation motif) that is also
found in the coreceptors of the B cell receptor
(BCR) (32). In the BCR, this motif mediates signal
transduction of cross-linked BCR by binding of
kinases, which are subsequently activated. Moreover, normal B cells strictly depend on BCR
expression for survival, and the ITAM motif is
essential also for this tonic survival signal (33, 34).
There is evidence that LMP2a is capable of mimicking the presence of a functional BCR, because in
LMP2a transgenic mouse models, B cells expressing
LMP2a but lacking a functional BCR are generated
in the bone marrow and are able to survive in the
periphery (35, 36).
Based on the features of LMP1 and LMP2a
discussed above, it has been proposed that these
viral proteins could have a decisive role in the
development of EBV-positive HL: by mimicking
the main survival signals of GC B cells CD40
signalling and Ig-crosslinking LMP1 and LMP2a
could allow the survival of GC B cells that would
otherwise undergo apoptosis (GC B cells die as a
28

default program by apoptosis, if they are not

rescued from cell death by survival signals such as
the ones mentioned above) (37). While this hypothesis may well explain the role EBV plays in the
pathogenesis of EBV-positive HL, it is less clear
whether LMP2a can have the same role also in the
established HRS cell clone. As is discussed in more
detail below, gene expression proling studies
showed that HRS cells have lost expression of
many B cell-specic genes (12). These molecules
include the Syk tyrosine kinase and the intracellular
adaptor protein SLP-65 (BLNK), two important
factors for BCR signalling. Since these molecules
appear to be essential for the function of LMP2a as
a BCR surrogate (38, 39), it is questionable whether
LMP2a can replace the BCR survival signal in the
established HRS cell clone via the conventional
BCR signalling pathway.
Another issue regarding the role of EBV in the
pathogenesis of HL relates to the question at which
step in the pathogenesis of HL HRS cell precursors
become infected by EBV. It has been proposed that
in healthy virus carriers, EBV predominantly infects
naive B cells and that (some of) these cells undergo
GC reactions to gain access to the memory B cell
compartment, the long-term reservoir of EBV (40).
Thus, according to this scenario, EBV would be
present already in a B cell that is driven into a GC
reaction and that after acquisition of additional
transforming events gives rise to a malignant HRS
cell clone. However, a study of the viral strategies
during infectious mononucleosis, the acute primary
EBV infection, suggested that also GC and or
memory B cells can be directly infected (41, 42).
While it is normally not possible to identify the
dierentiation stage at which an HRS cell precursor
was infected by EBV, we recently encountered a
composite mantle cell and classical HL in which only
a fraction of HRS cells was EBV-infected (Table 1)
(19). Interestingly, the EBV-positive and -negative
HRS cells carried not only shared somatic mutations
in the rearranged Ig V genes, but also a few
mutations that were found only in the EBV+
subclone. This suggests that the EBV infection
happened in a member of a mutating GC B cell
clone and that this cell as well as a non-infected other
member of the clone later gave rise to the HRS
tumour clone (presumably, the common precursor
of the two HRS subclones already carried a shared
transforming event). The alternative scenario,
namely that EBV was lost from an originally
homogenously EBV-infected HRS cell clone is
unlikely due to the fact that the EBV) HRS cell
subclone carried less somatic mutations than the
EBV+ subclone. Thus, this case is the rst example
for a HL case with indication that EBV infection
happened in a GC B cell, suggesting that at least in a

Pathogenesis of Hodgkins lymphoma

fraction of cases of EBV-positive HL the viral
infection may happen in GC and not in naive B cells.
The lost B cell identity of HRS cells

For the identication of genes dierentially

expressed between HRS cells and normal B cells
we performed two dierent approaches. In one
experiment, global gene expression of the HRS cell
line L1236 was compared to the expression prole
of ow cytometrically isolated tonsillar GC centroblasts using the serial analysis of gene expression (SAGE) method (11). In a second experiment,
gene expression proles of four HL-derived cell
lines (L428, L1236, KMH2 and HDLM2) were
compared to the proles of four normal B cell
subsets, namely naive B cells, memory B cells,
centroblasts (CD77-positive proliferating GC B
cells) and centrocytes (CD77-negative resting GC
B cells), using Aymetrix microarrays (10). One of
the observations made consistently in both types
of experiments was the detection of a global
down-regulation of most B cell-typical genes
(Fig. 1) (12). For a number of these genes, it
was already known that HRS cells usually lack
expression of these molecules, such as CD19,
CD20, CD79, immunoglobulin and the transcription factors Oct-2, Bob-1 and Pu-1 (4346).
However, the gene expression proling studies
identied many more genes that are normally
expressed by B cells and that are down-regulated
in HL cell lines, e.g. the kinases Syk, Lyn and Blk,
the adaptor molecule SLP-65, and the transcription factors Pu-B and A-myb (12). For several of
the molecules, we conrmed by immunohistochemistry that their down-regulation is not only a

feature of the HL lines, but is also characteristic

for HRS cells in HL biopsy specimens (12).
Interestingly, HRS cells retain expression of key
B cell-specic molecules important for B cell/T cell
interaction (CD80, CD86, MHC class II) and
surprisingly also of the B cell lineage commitment
and maintenance factor Pax-5 (Fig. 1) (47, 48).
What could be the cause for this global downregulation of B-lineage-specic genes? It has been
discussed that the HRS cell phenotype could reect
a plasma cell dierentiation, because plasma cells
down-regulate many B cell-lineage molecules, such
as CD19, CD20 and Blk, and HRS cells often
express molecules that are upregulated in plasma
cells (CD138, Mum-1) (49). However, the retained
expression of MHC class II and Pax-5, and in
particular the down-regulation of immunoglobulin
expression argue against this hypothesis. Based on
the puzzling observation that HRS cells usually
express the B lineage maintenance factor Pax-5, but
lack expression of several target genes of this
transcription factor, we speculated that Pax-5 may
be inactive because of somatic mutations. However,
no mutations were found in three HL lines that
were analysed for mutations in the Pax-5 gene (12).
Another possibility could be that the B cell
phenotype of the HRS cells is suppressed by a
dominant factor that is aberrantly expressed by
HRS cells. Indeed, it has been reported that HRS
cells express Notch-1, which plays an important
role in early lymphocyte development by suppressing a B cell dierentiation and supporting the
generation of T cells from lymphoid precursors
(50). Perhaps, the aberrant expression of Notch-1
by HRS cells contributes to the down-regulation of
B cell markers in these cells.

Ig receptor
CD79a and b

Downregulation of
many B-lineagespecific genes

CD40

Lyn
SLP-65

CD19

Retained expression of
Pax-5 and molecules
involved in antigenpresentation
MHC class II

Blk

CD80

CD20
Oct-2 PU.B

Syk

CD180

CD86
Pax-5

A-myb

fascin

MUM-1

Expression of plasma
cell-associated molecules

Nucleus

GATA-3
Notch-1

CD138

TARC
CD15

Aberrant expression of
molecules not expressed
by normal B cells

Fig. 1. The peculiar phenotype of HRS cells. The HRS cells in classical HL have a phenotype that does not resemble any
normal hematopoietic cell, and is very much dierent from the phenotype of its normal counterpart, i.e. GC B cells. Shown
are examples for B-lineage-specic genes down-regulated by HRS cells, plasma cell markers expressed by HRS cells, markers
of other lineages aberrantly expressed by HRS cells (TARC: dendritic cells, GATA-3: T cells, Notch-1: T cells, fascin:
dendritic and myeloid cells; CD15: myeloid cells) and B lineage genes that show retained expression in HRS cells. The
down-regulation of B cell markers is indicated by x through the molecules.

29

Kuppers et al.
Independent of the mechanism that causes the
global loss of the B cell identity of HRS cells, it is
an intriguing question whether this dramatic phenotypic change could be of advantage for the tumour
cells and may hence be selected for during the
pathogenesis of HL. On the background of the
proposed origin of HRS cells in classical HL from
pre-apoptotic GC B cells which failed to express a
high-anity BCR due to accumulation of disadvantageous V gene mutations, the loss of the B cell
identity could indeed represent a strategy of the
cells to escape from the stringent selection for
expression of an appropriate BCR (a high anity
BCR in the case of GC B cells), that governs the life
of a B cell, as discussed above.
Expression of receptor tyrosine kinases in HRS cells

Receptor tyrosine kinases (RTK) are frequently

involved in cellular transformation (51). Nevertheless, analysis of RTK expression in HRS cells has
so far been limited to a few studies of individual
RTKs, namely KIT, MET, HER2 and PDGFRA
(5258). The reported expression of HER2 and
KIT in HRS cells could, however, not be conrmed
by other groups and thus remains controversial
(52, 55). The MET can be detected in HRS cells and

also normal GC B cells with immunohistochemistry

(57), implying that expression in HRS cells is not
aberrant but likely due to the GC B cell origin of
the HRS cells.
For a more comprehensive analysis of RTK
expression we screened our global RNA expression
data of four HL cell lines (10), where 50 of 57
known RTKs were represented by probes, for
aberrant HRS cell-specic RTK expression. Eight
RTKs, which were aberrantly expressed in at least
two of the HRS cell lines were identied, and for
most of these RTKs we conrmed the RNA
expression data in Western blot analysis (59).
The expression of six of these RTKs was then
analysed in primary cases of classical HL and also
other B cell lymphomas. Immunohistochemistry
revealed that the RTKs PDGFRA, DDR2,
EPHB1, RON, TRKB and TRKA, were each
expressed in HRS cells of 3075% of cases
(Fig. 2A). None of these RTKs was expressed in
normal B cells. In B-NHL (MCL, FL, chronic
lymphocytic leukemia, Burkitt lymphoma and
diuse large B cell lymphoma) expression was only
occasionally observed with the most frequently
expressed RTK found only in 7% of cases. The
HRS cell-specic expression was further stressed by
analysis of two composite FL/HL cases, where

Fig. 2. Expression and activation of

the PDGFRA receptor tyrosine kinase
in HRS cells. Immunohistochemistry
using alkaline phosphatase with Fast
Red as substrate for PDGFRA (A)
revealed that PDGFRA is expressed in
the HRS cells of most classical HL
cases. A large fraction of these cases
coexpressed the PDGFRA high anity
ligand PDGFA (B), and activated,
phosphorylated PDGFRA (C) could
be demonstrated in a fraction of cases.
Immunohistochemistry with a panphospho-tyrosine specic antibody
using horseradish peroxidase with
DAB as substrate revealed aberrantly
high phospho-tyrosine contents in
HRS cells of a large fraction of HL
cases (D). The pan-phospho-tyrosine
stainings were comparable to tumours,
where activated receptor tyrosine kinases have important roles in cellular
transformation, like ALK-positive
anaplastic large cell lymphoma.

30

Pathogenesis of Hodgkins lymphoma

PDGFRA expression was restricted to the HRS
cells.
In the vast majority of cases at least one and in
most cases several RTKs were coexpressed. Aberrant RTK expression was more pronounced in
nodular sclerosis subtype. Here on average, nearly
four RTKs per case were found to be expressed,
while in mixed cellularity subtype an average below
two RTKs per case was observed. In lymphocytepredominant HL on average only one RTK was
expressed per case and the most frequently
expressed RTK was found in only 30% of cases.
Activated RTKs are phosphorylated on several
intracellular tyrosine residues and therefore we
used antibodies specic for phosphorylated RTKs
to analyse if they were not only aberrantly expressed
but also activated. For PDGFRA and TRKA/B we
could exemplarily show activation in considerable
fractions of cases (Fig. 2C). In addition, antibodies
detecting phospho-tyrosine in several proteins
revealed that the HRS cells in many cases had
elevated cellular phospho-tyrosine contents, comparable to other tumours, where activated RTKs
play important roles in pathogenesis (Fig. 2D).
Constitutive TK activities in tumours are frequently due to mutations in the TKs. However,
upon sequencing of the cDNAs of the RTKs under
investigation from the HL cell lines, we found no
activating mutations. As RTK activation could also
be due to presence of the ligands in the HL
inltrate, we performed immunohistochemistry for
the four RTK ligands for which suitable antibodies
were available. We indeed found that the DDR2
ligand collagen type I and the TRKA ligand, NGF
were present in several cases analysed; collagen type
I in the sclerotic bands branching in the tumour
inltrate and NGF in granulocytes, respectively.
This likely means, that these two RTKs can be
activated in paracrine fashions in a considerable
fraction of cases. For the PDGFRA and EPHB1
ligands PDGFA and EphrinB1, respectively, we
observed expression in HRS cells in a number of
cases (Fig. 2B). In most of the cases expressing
PDGFA or EphrinB1 in the HRS cells, the HRS
cells expressed also the corresponding RTK, suggesting autocrine activation in these cases.
Taken together, our analysis revealed aberrant
expression and activation of several dierent RTKs
in HRS cells of classical HL, most pronounced in
nodular sclerosis HL, and at least for four RTKs
activation seems to occur predominantly by their
ligands in paracrine or autocrine fashions.
Although no genetic changes causing deregulated
RTK activity were identied, the unprecedented
coexpression of multiple RTKs in a tumour
together with the observed aberrant expression in
the vast majority of cases indicates that aberrant

RTK activities contribute to HL pathogenesis.

These ndings indicate that TK inhibitors, e.g.
the PDGFRA inhibitor Imatinib, which is already
in clinical use, may be potential therapeutics for
classical HL, especially for treatment of nodular
sclerosis HL.
Expression profiling of microdissected HRS cells

Since HL cell lines most likely do not retain the

features of primary HRS cells in all important
aspects, and since the rare HL lines may not be
representative of typical primary HRS cells, as
discussed above, we plan to study dierential gene
expression of microdissected HRS cells in comparison with various B-NHL, the main normal B cell
populations and HL cell lines using gene expression
proling. Because of the rareness of the HRS cells in
the tumour tissue there was the need to establish a
reproducible and reliable amplication protocol
for RNA from lasermicrodissected HRS cells. Different RNA isolation methods including cell lysis
by detergent, two guanidine isothiocyanate-based
methods and a salt precipitation method were tested
for RNA yield and quality. Best results were
obtained with a kit based on salt precipitation
(Gentra, Minneapolis, MN, USA). The T7 RNA
polymerase-based RiboAmpTM RNA Amplication Kit (Arcturus, Mountain View, CA, USA) was
used with slight modications to amplify the RNA of
5001000 lasermicrodissected or FACS-sorted cells
by in vitro transcription. The in vitro transcribed
RNA was then converted into cDNA and the
double-stranded cDNA was subjected to the BioArray High Yield RNA Transcription Labelling Kit
(ENZO, Farmingdale, NY, USA) in a modied
manner. To test the reproducibility and reliability of
the procedure total RNA of three independent
replicates of 106 cells, 5 ng of diluted RNA and 500
FACS-sorted cells, all from the HL cell line L428,
were amplied, labelled and hybridised to Aymetrix genechip microarrays. Array data were analysed
according to the Aymetrix manual. All comparisons showed high reproducibility in terms of signal
correlation, discrepant calls and other quality criteria. The protocols were then applied to lasermicrodissected cells of primary HL, other B cell
lymphomas and FACS-sorted normal B cells subsets
and HL cell lines. A rst preliminary analysis of the
data by unsupervised hierarchical clustering shows
that the clustering is independent from the cell
isolation method. The two main branches of the
dendrogram subdivide the samples into normal
B cells and lymphoma cells. Among the normal
B cell subsets, there are three main subbranches
separating plasma cells, resting peripheral blood cells
(naive and memory B cells) and GC B cells (centro31

Kuppers et al.
blasts and centrocytes). In the branch of the lymphoma cells, the HL cell lines build a cluster of their
own, separate from the primary HL cases. The
preliminary data of the few lymphomas analysed so
far indicate that, as expected, there are apparently
signicant dierences between the HL cell lines and
primary HRS cells. Interestingly, HRS cells of
classical and lymphocyte predominant HL cluster
close to each other.
Acknowledgements

11.

12.

13.

The work discussed in this review was supported in part by

Grants from the Deutsche Forschungsgemeinschaft (Ku1315/
2-1, Br1238/6-1) and the Deutsche Krebshilfe, Mildred ScheelStiftung (70-3173-Tr3).

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