Cancer Genetics and Cytogenetics 186 (2008) 110e114

Short communication

BCL2 and BCL3 are recurrent translocation partners

of the IGH locus
Natalia Szymanowskaa,b, Wolfram Klapperc, Stefan Geska, Ralf Küppersd,
José I. Martı́n-Suberoa, Reiner Sieberta,*
a
Institute of Human Genetics, University Hospital Schleswig-Holstein, Campus Kiel, Christian-Albrechts
University, Schwanenweg 24, 24105 Kiel, Germany
b
Department of Clinical Chemistry and Molecular Diagnostics, Poznan University of Medical Sciences, Poznan, Poland
c
Department of Pathology, Hematopathology Section and Lymph Node Registry, University Hospital Schleswig-Holstein,
Campus Kiel, Christian-Albrechts University, Kiel, Germany
d
Institute for Cell Biology (Tumor Research), University of Duisburg-Essen, Essen, Germany
Received 16 May 2008; accepted 13 June 2008

Abstract Chromosomal translocations affecting the immunoglobulin heavy chain (IGH ) locus in chromo-
somal band 14q32 are the most frequent cytogenetic changes in B-cell lymphomas. We studied the
presence of IGH translocations in a consecutively ascertained series of 94 classical Hodgkin lympho-
mas (cHL) by combined immunofluorescence for CD30 and interphase cytogenetics (FICTION tech-
nique). The Hodgkin and ReedeSternberg cells of a total of 11 of 87 evaluable cases (13%) showed
signal patterns indicative of IGH translocations. To identify the translocation partners, these cases
were further studied with probes for the MYC, BCL2, BCL6, BCL3, REL/BCL11A, JAK2/PDCD1LG2
(alias PDL2) C14orf43, and C2TA loci. The IGH translocation partner could be identified in four cHL
and involved BCL2 and BCL3 in two cases each. Immunohistochemistry in cases with suitable ma-
terial revealed that tumor cells of the two cHL with IGH/BCL2 fusion and the cHL with IGH/BCL3
fusion expressed the BCL2 and BCL3 protein, respectively. These data indicate that BCL2 or BCL3
are recurrent translocation partners of the IGH locus in cHL; however, most of the translocation part-
ners of IGH translocations in cHL remain to be identified. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction hybridization (FISH) or combined immunofluorescence

and interface FISH (FICTION technique), recurrent genetic
Classical Hodgkin lymphoma (cHL) is an atypical B-cell
changes have been identified [7e10]. To date, the most com-
lymphoma in which the tumor cells, called Hodgkin and mon chromosomal changes associated with cHL are amplifi-
ReedeSternberg (HRS) cells, represent only 0.1e10% of
cations and gains of chromosomal regions 2p16 (REL and
the tumor biopsies, the rest being nontumoral cells derived
BCL11A genes) and 9p24 (JAK2) [7e9].
from an intense inflammatory reaction [1]. Conventional cy-
In B-cell malignancies other than cHL, chromosomal
togenetic studies from cHL biopsies are therefore very chal-
translocations affecting the immunoglobulin heavy chain
lenging and frequently render normal karyotypes [2e4]. The
(IGH ) locus, or its variants involving the light chain loci, rep-
few cases in which tumor-cell metaphases are obtained are
resent the most characteristic genetic changes [11e13].
characterized by highly unstable and complex karyotypes,
These translocations are assumed to arise as byproducts of
which frequently cannot be completely resolved [2e4]. an erroneous VDJ or switch recombination leading to an ille-
Breakpoints in several chromosomal regions, including
gitimate juxtaposition of the IGH locus to oncogenes, such as
14q32, have been reported [5,6]. Only with the help of molec-
BCL2, MYC, or CCND1, among many others [13]. As a result,
ular cytogenetic techniques targeting the HRS cells, such as
the physiological and highly regulated expression of these
conventional and array-based comparative genomic hybrid-
oncogenes becomes altered, which results in cell immortali-
ization from microdissected HRS cells, fluorescence in situ
zation and hyperproliferation [14].
* Corresponding author. Tel.: þ49-431-597-1774; fax: þ49-431-597- From the diagnostic point of view, different IGH trans-
1841. (R. Siebert). locations are associated with specific subtypes of B-cell
E-mail address: rsiebert@medgen.uni-kiel.de (R. Siebert). malignancies. For instance, the t(8;14)(q24;q32) (IGH/
0165-4608/08/$ e see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.cancergencyto.2008.06.007
 N. Szymanowska et al. / Cancer Genetics and Cytogenetics 186 (2008) 110e114 111

MYC ) is required to diagnose a Burkitt lymphoma [15] and immunofluorescence with commercially available break-
the t(14;18)(q32;q21) (IGH/BCL2) and t(11;14)(q13;q32) apart probes specific for the MYC, BCL2, and BCL6 loci
(IGH/CCND1) are present in the great majority of follicular (Abbott Molecular/Vysis, Des Plaines, IL) and home-
lymphomas and mantle cell lymphomas, respectively [13]. brewed break-apart probes specific for the BCL3, REL/
In the case of cHL, we recently described that ~20% of BCL11A, JAK2/PDCD1LG2 (alias PDL2), C14orf43, and
the cases contain chromosomal changes involving the IGH C2TA loci (clone information available in Martı́n-Subero
locus [16]. That study was based primarily on fluorescence et al. [16]). For BCL6, an additional commercial break-
in situ hybridization (FISH), however, and therefore only apart probe was also applied (Kreatech Biotechnology, Am-
cases with large HRS nuclei and hyperploid status could sterdam, The Netherlands). In each two cHLs with breaks
be evaluated. Also, case selection was biased toward cHL in the BCL2 and BCL3 loci, double-color double-fusion
submitted for cytogenetic diagnosis. In the present work, probes for IGH/BCL2 (Abbott Molecular/Vysis) and IGH/
to further identify and characterize IGH translocations in BCL3 (home-brewed) [19] were used to confirm fusion
cHL, we have studied an independent and unbiased series with the IGH locus.
of cHL cases by FICTION on cryosections. Slides were analyzed by use of Zeiss Axioskop 2 and
Zeiss Axio Imager A1 fluorescence microscopes (Zeiss,
Göttingen, Germany) equipped with appropriate filter sets
2. Materials and methods (AHF, Tübingen, Germany) and were documented using
the ISIS imaging system (Version 5.1, MetaSystems, Alt-
2.1. Patient material lussheim, Germany). The HRS cells were identified by
Cryostat sections of tumor biopsies from a total of 94 virtue of their typical morphology, hyperploid status, and
consecutively ascertained cHL patients were analyzed. expression of the CD30 antigen.
All the samples were obtained from the files of the Hema- The cutoff values for IGH break-apart probes have been
topathology Section (Christian-Albrechts University Kiel) described previously [20]; however, because FICTION al-
in the framework of the Molecular Mechanisms of Malig- lows the targeted analysis of HRS cells, cutoff values for
nant Lymphomas (MMML) network project, for which cen- FISH experiments are not applicable. To solve this issue,
tral and local ethics approval was obtained. All cases were 50 CD30-negative small cells served as internal controls
diagnosed as cHL according to the criteria of the World and were compared with the results obtained from at least
Health Organization [17]. Of the 94 cases, 89 could be fur- 10 HRS cells.
ther subtyped as mixed cellularity (n 5 49), nodular sclero-
sis (n 5 38), or lymphocyte depletion (n 5 2); the subtype 2.3. Immunohistochemical analysis for BCL2 and BCL3
was not available in 5 cases. The study population
comprised 51 men with a median age of 37 years (range, Immunohistochemistry was performed according to rou-
8e80) and 43 women with a median age of 30 years (range, tine protocols in paraffin-embedded material of cHLs show-
7e83). ing an IGH/BCL2 (n 5 2) or IGH/BCL3 (n 5 2) fusion
using antibodies against BCL2 (Zytopmed, Berlin, Ger-
many) and BCL3 (Leica, Wetzlar, Germany).
2.2. Combined immunofluorescence and interphase
cytogenetics
A molecular cytogenetic analysis combining fluores- 3. Results and discussion
cence immunophenotyping and interphase FISH (FICTION
technique) was performed on cryosections as previously Cryosections from a total of 94 cHL biopsies were stud-
described [18], using a mouse monoclonal antibody to ied with a FICTION assay aimed at detecting chromosomal
CD30 (DakoCytomation, Hamburg, Germany) detected breakpoints in the IGH locus. Sufficient large CD30-posi-
with a rabbit anti-mouse secondary antibody conjugated tive HRS cells were identified in 87 cHLs (93%); 7 cases
with Alexa Fluor 594 (Molecular Probes, Leiden, The were not evaluable, either for lack of clearly recognizable
Netherlands) and a triple-color hybridization probe specific HRS cells or because of insufficient hybridization quality.
for the IGH locus. This probe was made of the locus-spe- The HRS cells from 11 of the 87 evaluable cHLs (13%)
cific identifier (LSI) break-apart IGH FISH probe (labeled displayed signal patterns indicative of a breakpoint in the
with Vysis SpectrumOrange and SpectrumGreen; Abbott IGH locus (Table 1), whereas the bystander cells regularly
Molecular, Des Plaines, IL) in combination with bacterial showed a normal signal constellation. In all these cases,
artificial chromosome (BAC) clone RP11-417P24 labeled there was no evidence for a coexisting or previous B-cell
with diethyl aminomethyl coumarin [16]. non-Hodgkin lymphoma. From these 11 cHLs, hybridiza-
Classical HL samples showing a signal constellation tion signals of the BAC clone RP11-417P24 were reduced
indicative of a chromosomal breakpoint in the IGH locus or absent in the HRS cells of 10 cases, compared with those
were further analyzed to identify the translocation partner. in the small bystander cells. This pattern, which was also
To this aim, FICTION was performed combining CD30 observed in the previous cHL series studied for IGH
112 N. Szymanowska et al. / Cancer Genetics and Cytogenetics 186 (2008) 110e114

Table 1
Findings from FICTION analyses aimed at detecting the IGH translocation partner in 11 cases of classic Hodgkin lymphoma in which HRS cells showed
a chromosomal breakpoint in the IGH locus
REL/ JAK2/
IGH MYC BCL6 BCL2 BCL3 BCL11A PDCD1LG2 C2TA C14orf43 Translocation
No. (14q32)a (8q24)b (3q27)b,c (18q21)b (19q13)a (2p16)a (9p24)a,d (16p13)a (14q24)a IGH/BCL2b IGH/BCL3a partner
1 þ  /  þ     ND þ BCL3
2 þ  / þ      þ ND BCL2
3 þ NE /       ND ND d
4 þ  /       ND ND d
5 þ  /       ND ND d
6 þ  /     NE  ND ND d
7 þ  /       ND ND d
8 þ  /       ND ND d
9 þ  / þ      þ ND BCL2
10 þ  /  þ     ND þ BCL3
11 þ  /       ND ND d
Abbreviations: ND, not done; NE, not evaluable; FICTION, fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation
of neoplasms [18]; HRS, Hodgkin and ReedeSternberg; þ or , signal constellation indicative of the presence (þ) or absence () of a chromosomal break-
point or gene fusion in HRS cells.
a
Our own home-brewed FISH probe [16,19].
b
Commercially available FISH probe (AbbotteVysis or Kreatech);
c
Two results are given, separated by a slash mark: for the AbbotteVysis probe first, then the Kreatech probe.
d
Alias PDL2.

translocations [16], points to the presence of deletions af- previously demonstrated by FISH in only a single case re-
fecting part of the IGH constant segments. In turn, this find- port, by Miura et al. [26]. The present identification of two
ing suggests that class switch recombination has taken additional cHLs in which HRS cells display an IGH/BCL2
place in these HRS cells, which is a well-known phenome- fusion indicates that this rearrangement is rare but never-
non established by molecular studies in cHL [21]. theless recurrent in cHL.
Analyzing the location of the RP11-417P24 signals with The translocation t(14;19)(q32;q13) leading to an IGH/
regard to the isolated red or green signals in cases with the BCL3 fusion is frequently associated with B-cell chronic
IGH split indicated that the breakpoints within the IGH locus lymphocytic leukemias [27]. We have recently reported,
in cHL are heterogeneous, confirming previous results [16]. however, that IG/BCL3 fusions do also appear in a wide va-
FICTION combining CD30 with probes for MYC, BCL2, riety of different mature B-cell malignancies [19]. The two
BCL6, BCL3, REL/BCL11A, JAK2/ PDCD1LG2, C14orf43, cHLs with an IGH/BCL3 fusion described here, together
and C2TA was applied to the 11 cHLs with chromosomal with one additional cHL previously identified [16,28], indi-
breakpoints in the IGH locus. Four cHLs showed signal cate that IGH/BCL3 fusion is also a recurrent chromosomal
constellations indicating the presence of breakpoints, which change in cHL.
affected BCL2 and BCL3 in two cases each (Table 1; Figs. The IGH locus is actively expressed in B-cells, and it is
1A and 1D). Juxtaposition of IGH and BCL2 or BCL3 was widely accepted that juxtaposition of oncogenes and IGH
confirmed in these cases using double-color double-fusion regulatory elements through chromosomal translocations
IGH/BCL2 and IGH/BCL3 probes (Figs. 1B and 1E). leads to overexpression of the former [13]. Because the
The translocation t(14;18)(q32;q21) leading to an IGH/ IGH locus is downregulated in the HRS cells of cHL
BCL2 fusion is the cytogenetic hallmark of follicular lym- [1,29,30], however, the pathological effect of IGH translo-
phomas [13]. In HL, several PCR studies reported in the cations in this disease remains unclear. The presence of
1990s suggested the presence of IGH/BCL2 fusions [22]. composite non-Hodgkin and Hodgkin lymphomas sharing
One of these studies showed that 11 of 28 HLs (39%) were the same IGH translocation suggests that oncogene deregu-
positive for IGH/BCL2 by PCR, but only one of those pa- lation by IGH translocations in cHL might represent
tients showed a t(14;18) by conventional cytogenetics [6]. a primary event fostering B-cell transformation [31]. Addi-
A subsequent study analyzing single HRS cells demon- tionally, it has been shown that an oncogene can be
strated that such IGH/BCL2 fusion was not present in the expressed in cHL from the allele juxtaposed to IGH, which
HRS cells, but rather in the normal bystander cells [23]. indicates that oncogene deregulation can be possible
In line with this finding, it has been demonstrated that pe- regardless of IGH silencing [31].
ripheral blood cells of ~50% of healthy individuals and pa- In the two cases with IGH/BCL2 fusion described here
tients with nonmalignant disease show the presence of IGH/ (cases 2 and 9), immunohistochemical analyses revealed
BCL2 rearrangements by PCR [24,25]. To our knowledge, that the BCL2 protein was expressed by most HRS cells
fusion of IGH and BCL2 in HRS cells of cHL (without in both cases (Fig. 1C). With regard to BCL3 expression
coexisting or previous follicular lymphoma) has been in the two cases with IGH/BCL3 fusion, the HRS cells of
 N. Szymanowska et al. / Cancer Genetics and Cytogenetics 186 (2008) 110e114 113

Fig. 1. Fluorescence immunophenotyping and interphase cytogenetic (FICTION) [18] and immunohistochemical analyses in classical Hodgkin lymphoma
patients with IGH translocations. (A) Case 9: A CD30-positive Hodgkin and ReedeSternberg (HRS) cell, displayed in blue, shows a split, with a BCL2
break-apart probe (arrows). (B) Case 2: Confirmation of BCL2 as a translocation partner of IGH in an HRS cell, with an IGH/BCL2 dual-color probe. Fusion
signals indicate the presence of an IGH/BCL2 juxtaposition (arrows). (C) Case 2: Immunohistochemical staining showing that the HRS cells express BCL2
(arrows). (D) Case 1: A CD30-positive HRS cell, displayed in blue, shows a split, with a BCL3 break-apart probe (arrows). To the right of this HRS cell is
a small CD30-negative cell displaying the normal signal constellation, two colocalized signals. (E) Case 1: Confirmation of BCL3 as the translocation partner
of IGH in an HRS cell using an IGH/BCL3 dual-color probe. Fusion signals indicate the presence of an IGH/BCL3 juxtaposition (arrows). (F) Case 10:
Immunohistochemical staining showing that the HRS cells in a case with an IGH/BCL3 fusion express BCL3 (arrows). FICTION figures are shown using
a false-color display.

one case (no. 10) expressed the BCL3 protein by immuno- Acknowledgments
histochemistry (Fig. 1F); the other case (no. 1) was not
evaluable, however, because of poor immunoreactivity of This study was supported by the Deutsche Krebshilfe
the paraffin-embedded tissue obtained at autopsy. The find- (Grant numbers 107748 and 107736). At the time of the
ing that these cHLs with IGH/BCL2 or IGH/BCL3 fusions research, Natalia Szymanowska was recipient of an
express BCL2 and BCL3, respectively, is not surprising, Erasmus/Socrates scholarship from the European Union.
because HRS cells in cHL frequently express these two We thank Claudia Becher, Dorit Schuster, Magret Ratjen,
proteins, as indicated by immunohistochemistry [32,33]. Reina Zühlke-Jenisch, and Edda Seweke-Wessel for their
These data argue for a role of BCL2 and BCL3 in the path- excellent technical assistance.
ogenesis of cHL, but a causal relation between IGH trans-
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