1. No category

From www.bloodjournal.org by guest on November 17, 2014. For personal use only.
1994 83: 223-230
The bcl-2 oncogene in Hodgkin's disease arising in the setting of
follicular non-Hodgkin's lymphoma
DP LeBrun, BY Ngan, LM Weiss, P Huie, RA Warnke and ML Cleary
Updated information and services can be found at:
http://www.bloodjournal.org/content/83/1/223.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
From www.bloodjournal.org by guest on November 17, 2014. For personal use only.
The bcl-2 Oncogene in Hodgkin’s Disease Arising in the Setting
of Follicular
Non-Hodgkin’s Lymphoma
By David P. LeBrun,Bo-Yee Ngan, Lawrence M. Weiss, Philip Huie, Roger A. Warnke, and Michael L. Cleary
Expression of
the bcl-2 proto-oncogeneon chromosome 81
is deregulated by the 14; 18chromosomal translocation,
an abnormality that is consistently associated with follicular non-Hodgkin’s lymphomas (NHL). Because bcl-2 is believed to function by prolongingcell survival rather than by
increasing proliferation, the presence of t(l4; 18) in Hodgkin’s disease (HD) would have profound implications for
the pathogenesisof this neoplasm. W e evaluated 32 cases
of HD for t(l4;18) by polymerase chain reaction (PCR).
These results were correlated with expression of bcl-2 oncogenic protein by Hodgkin cells and with thepresence of
Epstein-Barr virus (EBV), as determined by immunohistochemistry or in situ hybridization. PCR provided evidence
of t(l4; 18) in only 2 HD cases (6%),both of which were
associated with a prior historyof follicular lymphoma, and
both of which were among the 7 cases (22%)with strong
bcl-2 expression in Hodgkin cells. In atleast 1 of the cases,
the translocation involved identical chromosomal breakpoints in both types of lymphoma. Furthermore, 7 additional cases of combined follicular NHL and HD showed
strong bcl-2 staining in Hodgkin cells. Although EBV was
detected in 6 of 30 cases, it was not associated with
t(14; 18) and usually not with strong bcl-2 expression.
These results suggest that a small proportion ofHD cases
might evolve from follicular NHL, possibly through molecular events superimposed onthe t(l4; 18).High-level bcl-2
expression in Hodgkin cells is a potentially useful but not
definitive marker for these cases.
0 1994 by The AmericanSociety of Hematology.
T
scripts. But EBV was detected in only 1 of the cases with
enhanced bel-2 expression. Our findings lead us to propose
that, although the t( 14; 18)has a low prevalence in HD, the
small number of H D cases that do carry this translocation
may have arisen by “clonal evolution” from a previous follicular NHL.
HE PATHOGENESISOF Hodgkin’s disease (HD) and
the origin of the Reed-Sternberg cell and its variants
(Hodgkin cells), the neoplastic cells of HD, have been the
subject of much study and debate. Overthe years, granulocytes, histiocytes, and interdigitating reticulum cells have
been proposed as the cell of origin of Hodgkin cells, but
more prominent in the recent literature are immunophenotypic and molecular studies supporting a lymphoid origin. In light ofthese studies, the reported detection by several groups of the 14;18 translocation, a chromosomal
marker of follicular non-Hodgkin’s lymphoma (NHL),’ in
up to 32% of H D cases by the polymerase chain reaction
(PCR) is particularly p r o v ~ c a t i v e . ~ - ~
The t( 14; 18)(q32,q2 1) translocation deregulates expression of the bel-2 proto-oncogene, whose protein product is
believed to function by prolonging cell life rather than by
enhancing p r ~ l i f e r a t i o n . ’ ~Because
’~
confirmation of the
occurrence of the t( 14; 18) in a significant proportion of
cases would have profound implications for the pathogenetic mechanism of HD, we used PCR to evaluate 32 cases
of H D for this translocation. We observed that the cases
with detectable t( 14; 18) translocations were among those
with a prior history of follicular lymphoma. Immunohistochemistry in these cases showed enhanced bel-2 expression
that was localized to Reed-Sternberg cells and mononuclear
variants. PCR and sequence analysis of frozen biopsy tissue
from the H D and from the previous follicular lymphoma
was possible in 1 patient and showed involvement of identical chromosomal breakpoints by the t( 14; 18) in both diseases. Furthermore, evaluation of additional cases showed
that all but 1 of 1 I lymphomas in which HD andfollicular
lymphoma occurred in the same patient showed enhanced
bel-2 expression in the Hodgkin cells. However, not all bcl2 expressing H D cases contained detectable t( 14; 18)breakpoints or had a history of follicular lymphoma.
Because the Epstein-Barr virus (EBV) has been shown to
induce bel-2 expression in cultured cells by means of EBVencoded latent membrane protein-l (LMP-1),14 the presence of EBV was assessed by
immunostaining of H D tissues
for LMP- 1 or by in situ hybridization for EBV-specifictran-
Blood, Vol83, No 1 (January l ) , 1994: pp 223-230
MATERIALSANDMETHODS
Thirty-two cases of H D were selected from the files of the Laboratory of the Surgical Pathology at Stanford University (Stanford,
CA) on the basis of availability of frozen tissue or DNA and the
ccxesponding paraffin-tissue blocks or unstained paraffin sections.
Paraffin sections stained with hematoxylin and eosin were reviewed
from each case, and the cases were categorized according to the Rye
classification.15Of these cases, 4 were not classifiable; 2 met thecriteria of interfollicular HD,I6 and the remaining 2 cases had extranodal disease (gallbladder and lung) with pathologic findings diagnostic of H D but impossible to subclassify. Hospital charts were
reviewed for the 3 cases with prior follicular lymphoma, whereas
clinical information on the othercases was obtained from pathology
reports. A group of 8 additional cases was assembled using the concurrent or sequential occurrence of
HD and follicular lymphoma in
the same patientas selection criteria.
For the PCR, high molecular weight DNA was purified, using
From the Department of Pathology, Stanford University Medical
Center, Stanford; and the Department of Pathology, City of Hope
National Medical Center, Duarte, CA.
Submitted June 24, 1993; accepted September 3, 1993.
Supported in part by Grant Nos. CA 34233, 33119, 42971, and
50341 from the National Cancer Institute, National Institutes of
Health. D.P.L. and B.-Y.N. are Research Fellows of the Medical
Research Council ofCanada.
Address reprint requests to David P. LeBrun. MD, Department of
Pathology, Room L-212,Stanford University Medical Center, Stanford, CA 94305.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with18 U.S.C.section 1734 solelyto
indicate thisfact.
01994 by The American Society qf Hematology.
0006-4971/94/8301-0018$3.00/0
223
From www.bloodjournal.org by guest on November 17, 2014. For personal use only.
224
LEBRUN ET AL
techniques which have been previously described,” from snap-froZen biopsy specimens that had been stored at -70°C. Two micrograms of purified DNA was subjected to 30 cycles of amplification
by PCR using the Perkin-ElmerDNA thermal cycler, Taq polymerase, synthetic oligonucleotide primers MC4, MC5, and cellularity
MC8. and
reagents obtained commercially (Perkin Elmer-Cetus, Emeryville,
CA). Duplicate Southern blots were prepared, using one-fifth of the
reaction products for each, and hybridized with 32P-kinased synthetic oligonucleotides MC6 or MC12 as probes. Oligonucleotide
MC4, S-ACCTGAGGAGACGGTGACC-3’,is complementary to
a sequence common tothe six JH regions ofchromosome 14; MC5.
5’-TGCTGTGGTTGATATTTCGA-3‘,corresponds to a sequence
within the bcl-2 gene on chromosome18. immediately 5‘to the major breakpoint region (MBR), and is complementary to the negative
strand at that site: MC8, S-GACTCCTTTACGTGCTGGTACC3’, corresponds to a sequence immediately 5’ to the minor cluster
region (MCR) on chromosome 18: and MC6, T-GTATTTAGTTATGGCCTATACACTATTTGTGAGCAAAGGTG-3‘ and
MC 12, 5’-GATGGCTTTGCTGAGAGGTAT-3‘,correspond to
sequences within the anticipated PCR products from the MBR and
MCR, re~pectively.‘~.’~
DNA from 2 follicular lymphomas with
known t( 14; 18)’s, one involving the MBR and theother the MCR,
were run with each group of cases as positive controls. The minimum threshold of positivity was defined by control PCR samples
containing2 X
fig of positive control DNA diluted in 2 fig of
DNA from a reactive tonsil. The method used for genomic Southern blot analysis for t( 14; 18) has been previously described.20For
case HH, PCR products of appropriate size were excised from an
agarose gel and cloned into a plasmid vector (pCR 1000) using a
commercially-obtained kit (TA CloningKit: Invitrogen, San Diego,
CA) according to the instructions provided. Inserts were then sequenced using the Sequenase kit (US Biochemical, Cleveland, OH).
The monoclonal antibody (MoAb; clone no. 124) used for immunohistochemical detection of bcl-2 protein was provided by Dr
David Mason (Oxford University, Oxford, UK). The production
and characterization of this reagent, as well as theavidin-biotin detection method used in immunohistochemical staining, have been
previously described.2’,22Immunostained sections were examined
under high magnification using a standard light microscope. Cytoplasmic staining of Reed-Sternberg cells and variants was considered strong if it was clearly more intensethan in surrounding, nonneoplastic lymphocytes, weak if it was present but no more intense
than in lymphocytes, or absent. In these latter cases, the staining of
background lymphocytes served as an internal positive control.
This precaution was necessary because processing of tissue for paraffin embedding can impair detectability of the bcl-2 epitope by
MoAbs. In 25 cases in which additional snap-frozen tissue specimens were available, frozen section immunohistochemistry was
performed for detection of the EBV-associated protein LMP- 1 using a MoAb (CSI-4)provided by Drs L.S. Young and A.B. Rickinson (University of Birmingham,Birmingham, UK). Production
and characterization of this reagent have been previously described.23The immunostaining method used was similar to that
used to detect bcl-2, except that streptavidin-conjugated horseradish peroxidase and diaminobenzidine were replaced by alkaline
phosphatase and Fast Red, respectively, to avoid difficulties in interpretation caused by endogenous peroxidase in eosinophils. In
situ hybridization to detect EBV-specific EBER-I region transcripts
was performed on 27 cases from which a sufficient number of unstained paraffin sections were available. The technique used has
been described in detail el~ewhere.’~
RESULTS
The results of PCR analysis of H D tissues for t( 14; 18) are
shown in Table 1. Of 32 evaluated cases, 2 (6%)contained
Table 1. PCR Detection of t(14;18)
HD Subtype
Lymphocyte
predominance
Mixed
Nodular sclerosis
Unclassified
Total
Cases Evaluated
3
8
Cases Wlth 1114 18)
17
0
1
1
4
0
32
2 (6%)
amplifiable t( 14; 18) products, both of which hybridized
with a probe to the MBR. Both HH and HN, as well as a
third case, VS, without at( 14; 18), had a history of prior follicular lymphoma. Clinical details of these 3 cases are presented in Table 2. DNA from a snap-frozen biopsy specimen available from the follicular large-cell lymphoma of
HH was examined by 14; 18 PCR, which showed products
of identical size to those from this patient’s HD specimen
(Fig 1). At least two bands are detected from these specimens because of priming by the consensus JH oligonucleotide at complimentary sites in contiguous JH genes. Genomic Southernblotting of the HH DNA specimens and
hybridization with a DNA probe for the bcl-2 MBR (PFL1)” showed rearranged and germline bands in the follicular
lymphoma and only a germline band in the H D specimen.
The latter result was consistent with the presence oft( 14; 18)
carrying cells in the HD specimen below the threshold of
detection by Southern blot (1%). The presence of the same
breakpoints in both lymphomas in case HH was confirmed
by DNA sequencing (Fig 2). The relative abundance of the
cells containing t( 14;18) in the analyzed follicular lymphoma andH D tissue from HH was determined by dilution
analysis of the DNA purified from these specimens (Fig 3 ) .
This indicated that fewer than 1% of the cells in the HD
tissue from HH contained bel-2-IgH fusion, a result consistent with the estimated abundance of Hodgkin cells as determined by morphologic and immunologic studies (see below) and by Southern blot analysis.
The immunohistochemical staining results are shown in
Tables 3 and 4. Cytoplasmic bcl-2 staining was present in
Hodgkin cells of 20 cases (63%). However, strong staining
was present in only 7 cases (22%),including the 3 cases with
previous follicular lymphoma (Fig 4). Thus, both cases in
which a t( 14; 18) was detectable were among those with
strong bcl-2 expression, an association of marginal statistical
significance (P = .04). In the 3 cases with previous follicular
lymphoma, enhanced bel-2 expression was confined to the
Hodgkin cells in contrast with the small lymphocytes that
showed only background level staining. By these criteria, no
NHL cells appeared to be present in the analyzed tissue sections. No t( 14; 18) was detected in any of the cases in which
bel-2 expression was weak or absent. LMP-l was detected
by immunostaining in 4 of 25 cases (16%), whereas in situ
hybridization detected EBV-specific messenger RNA in 6 of
27 cases (22%; Table 4). Twenty-two cases were studied by
both methods with complete concordance in the results
(EBV detected in the same4 cases by either method).Therefore, the results obtained by the two methods were consid-
From www.bloodjournal.org by guest on November 17, 2014. For personal use only.
bcl-2 AND
225
Table 2. Cases With Follicular Lymphoma PrecedingHD
Patient
Initials
Lymphoma
TypeJDate
HH
HN
FLC/1970
FSC/1976
FSC/1982
vs
Treatment
Lymphoid irradiation
Lymphoid irradiation
Chemotherapy
NS/1985
MC11987
NS/1987
t(14;18)
in HD Tissue
Staining for
BCL-P
Yes
Yes
No
Strong
Strong
Strong
Abbreviations: FLC, follicular large cell;FSC, follicular small cleaved cell; NS, nodular sclerosing; MC, mixed cellularity.
ered together (EBV present in 6 of 30 cases) and showed no
clear relationship between EBV status and enhanced hcl-2
expression.
The results prompted a retrospective immunohistochemical analysis of 8 additional cases in which HD andfollicular
NHL occurred in the samepatient (Table5 ) . Strong staining
of Hodgkin cells for hcl-2 was present in all but 1 of these
cases (staining of small lymphocytes, used as an internal
positive control, was also absent in this case making the result uninterpretable), and the association between strong
hcl-2 expression in Hodgkin cells and prior or concurrent
follicular lymphoma was statistically significant (P< .OO l ) .
The results of diagnostic immunophenotyping supported
the diagnosis of HD in each case. Molecular studies were
Y
Y
23.1 kb
1057 bp
770 bp
-W
+
612bp +
498bp -W
9.4 kb
PCR
Southern
Fig 1. PCR and Southern blot analysis of the t(14: 18)
chromosomal translocation in follicular lymphoma and HD biopsy specimens from patient HH are shown. Snap-frozenbiopsy specimensof
both follicular lymphoma and HD tissues were available from patient HH. DNA was purified from these and subjected to PCR and
genomic Southern blot analysis. Theidentical size of PCR products
from the two specimens strongly suggests involvement of the same
chromosomal breakpoints in the two lymphoma types. The genomic Southern blot of the same DNA digested with the restriction
enzyme 8arnHl and hybridized with the PFL-1 probe for the M8R
on chromosome 18 showed an approximately 18-kbgermline band
and an approximately 13-kb revisible in both samples (-)
arranged band that isvisible in thefollicular lymphoma specimen
only (small arrow). The relative abundance of neoplastic cells in the
HD specimen is probably too low for detection by Southern blotting
without amplification.
not performed on these additional cases because of a lack of
appropriately matched frozen tissues or theobvious coexistence of NHL andHodgkin cells in the samespecimens.
DISCUSSION
The Reed-Sternberg cell and its variants are recognized as
the neoplastic component of the mixed population of hematolymphoid cells generally present in biopsy specimens
of HD tissue. Although they have long been presumed to
have arisen from a hematolymphoid precursor? study of
Hodgkin cells at the molecular level has been hampered by
their relative paucity in tissues involved by the disease.
These cells generally comprise less than I % of cells present
in such tissues, whereas detection by traditional Southern
blotting methods requires the DNA of interest to comprise
at least 1% of the total DNA present in a sample.” With its
ability to amplify specific DNA sequences present in as few
as 1 in IO5 cells,” the PCR is potentially well suited to the
study of HD. Furthermore, the tight clustering of breakpoints on chromosome I8 and in the JH genes on chromosome 14 have permitted us and others to design synthetic
oligonucleotide primers for PCR complementary to sites
that flank the chromosomal breakpointsinvolved in the vast
majority of 14: 18 trans location^.'^^'^-^^
Our results from applying the PCR to 32 unselected cases
of HD indicate that the 14; 18 translocation occurs infrequently in this neoplasm (6%).When t( 14: 18) does occurin
HD, it appears to be restricted to cases associated with a
previous follicular NHL. The translocation was detected in
2 of the 3 cases with such an association in our series. In I of
these cases, the size and nucleotide sequence of PCR products from the HDtissue and theolder follicular lymphoma
tissue were identical, indicating involvement in the two neoplasms of the same breakpoints on chromosomes 14 and
18 and suggesting clonal evolution of HD from follicular
lymphoma.
Alternatively, the presence ofa minimal residual quantity
of follicular lymphoma in the HD tissue could account for
5’- AAT GCA GTG TGC ?TA CTT GCA CTA CGG TAT -3’
Chromosome
Insertion
18
Chromosome
14
Fig 2. DNA sequence analysis of t(l4; 18)breakpoint from case
HH is shown. The samebreakpointson chromosomes 18 and 14as
well as an identical 11-nucleotide insertion (underlined)were identified in the PCR products from this patient’s follicular lymphoma
and HD specimens.
From www.bloodjournal.org by guest on November 17, 2014. For personal use only.
LEBRUNETAL
226
Dilution of Sample DNA
cy
r(
z
z
c4
W
z
v)
z
z
Or -
Follicular
Cell
Large
Hodgkin's
nonneoplastic tonsillar DNA, and 2 pg of total
DNA from each dilution was subjected to PCR
amplification. Whereas the t(l4; 18) detectable
is
in thefollicular lymphoma DNA after dilution to 1
in 104,as indicated by a faint hybridization signal,
no hybridization signal is present from the HD
DNA beyond a dilution of 1 in 10'. indicating an
approximately 100-fold lower abundance of
t(l4; 18)-containingcells in the HD specimen relative to the follicular large-cell lymphoma. Because neoplastic cells constituted 50% to 75% of
cells in thefollicular lymphoma specimen as determined morphologically and immunologically,
this result confirms an abundance of t(l4; 18)containing cells in the HD tissue of lessthan 1%.
0
these results. Although we have not excluded this possibility
rigorously, several findings make it unlikely. In our series.
strong he/-2 expression seems to be correlated with the presence oft( 14; 18) (2 of 2 v 5 of 30, P = .04) and is strongly
correlated with the co-occurrence of follicular lymphoma
(IO of IO cases v 4 of 29, P < .OOl). Therefore, the clear
localization to Hodgkin cells of abundant he/-2 protein in
both cases with detectable t( 14: 18) makes these cells the
most likely site for the translocation. Furthermore, PCR
amplification of serially diluted DNA specimens from one
of the cases indicates the abundanceof translocation-carrying cells to be approximately 0.5'70, a quantity that is in
agreement with morphologic and immunologic evaluation
of the number of Reed-Sternberg cells and variants in the
HD tissue from this case. Finally, no trace of follicular lymphoma was detectable morphologically or immunologically
in the HD tissue from either of the t( 14; 18)-associated cases.
The term "composite lymphoma" refers to a tumor in
which two distinct histologic subtypes of lymphoma are
present in the same mass.3o Three large series of composite
lymphomas include a total of 37 casescomposed of HD and
NHL.3"32 In I5 of these (41%) the NHL component was
follicular, and the diagnosis of follicular lymphoma is noted
to have antedated that of HD in 3 cases. These studies must
be distinguished from studies of NHL that follow therapy
for HD. In this setting, the most common secondary malignancy is acute myeloblastic leukemia.33However NHL may
also occur in this setting and are most often diffuse, histoIogicIy aggressive types.34
It is also noteworthy that follicular lymphoma preceded
HD in each ofthe 6 cases in our extended series in which the
diseases were diagnosed on separate occasions. Of relevance
to this observation is a recent study by Zarate-Osorno et a13'
describing9 cases of HD after NHL. The NHL was follicular
in 7 cases (78%),although this prevalence decreased to 14 of
38 cases (37%) when additional cases from the literature
were considered.
Our findings and those in the literature suggest that a
small subset of lymphomas thatmeets the morphologic and
immunologic criteria of HD has evolved from a preexisting
follicular NHL. Although the low prevalence oft( 14; 18) in
our initial group of HD cases indicates that such a progression does not accountfor the preponderance of HD, follicular lymphoma may represent merely one process among
Table 4. Correlation of h/-2 Expression
With t(l4;18)and EBV Status
Table 3. Immunohistochemical Detection
of k l - 2 ExDressionin Hodakin Cells
bcl-2 Staining
t(14;lS)Present
bcl-2Staining
HD Subtype
3
Strong
1
Lymphocyte predominance
Mixed cellularity
3
Nodular sclerosis
Unclassified
0
Total
7 (22%)
Weak
Fig 3. Relative abundance of t(14; 18)-containing cells by dilution analysis of DNA from follicular large-cell and HDspecimens is shown.
DNA from thefollicular large-cell lymphoma and
HD specimens from HH was serially diluted in
Absent
2
EBV Detectable in HodgkinCells'
Strong
Weak
1/12
Absent
217
0113
0112
1l 6
4112
Total
2/32 (6%)
6/30 (20%)
2
5
0
7
1
13 (41%)
5
3
12 (37%)
Values are the number of cases positive/number of cases evaluated.
*Refers to EBV detectabilitybyeitherimmunohistochemistryfor
LMP-1 or in situ hybridization Efor
BER transcripts.
From www.bloodjournal.org by guest on November 17, 2014. For personal use only.
bcl-2 ANDHODGKIN‘SDISEASE
Fig 4. Immunohistochemicaldetectionof bcl-2 expressionin
paraffin-embedded HD tissue from patient HH is shown. Note intense stainingfor k / - 2 protein in Reed-Sternberg cells and mononuclearvariants relative to small,“background“lymphocytes
(anti-bcl-2; original magnification X 200).
several that predisposes to the later developmentof HD. A
common feature of such processes may be the ability to produce a pool of inappropriately long-lived lymphoid cells.
These cells may then constitute “fertile soil” for the occurrence of superimposed molecular events specific to HD.It is
these latter molecular events, so far unidentified, that may
enhance the proliferative activity of affected cells and bring
about the emergence of a more aggressive neoplastic subclone. The samegenetic alterations may also give riseto the
constellation of features, such as a polymorphous background of host cells and the acquisition by neoplastic cells
ofan immunophenotypesimilar to that ofan
activated lymp h ~ c y t ewhich
, ~ ~ are currently used to distinguish H D from
other hematolymphoid neoplasms. This process may be
analogous to the superimposition of c-myc activation on a
preexisting t( 14; 18)that has been documented to be associated clinically with progression from follicular to diffuse
lymphoblastic lymphoma37 and experimentally with progression from polyclonal follicular hyperplasia to high-grade
malignant lymphoma in t( 14; I8)-canying transgenic
mice.38In addition, the association of H D with chronic lymphocytic l e ~ k e m i a ~and
~ , ~ ’the rare association of H D with
mycosis f ~ n g o i d e s ~imply
’ . ~ ~that similar mechanisms may
be involved in the evolution o f H D from several other indolent lymphoproliferative diseases.
B cells infected by EBV represent an attractive candidate
for a Hodgkin-cell precursor. EBV infection is known to
227
lead to a circulating population of long-lived B-lineage lymp h o c y t e ~ .Furthermore,
~~
EBV-specific nucleic acid sequences and proteins have been detected in H D cells in up
to 48% of cases, with some studies indicatingan especially
high EBV prevalence in the mixed cellularity
Recent evidence indicates that prolongation of B-cell survival in vitro by EBV is related to induction of hcl-2expression by virus-encoded LMP-1.I4 Interestingly, the EBV genome itself contains a gene that codesfor BHRFI, a
hypothetical protein of unknownfunction that has sequence homology to h ~ l - 2 . ‘ ~
In the current study, the
possibility that induction by EBV
may account for some cases in which bel-2 expression was
not associated with a detectable t( 14; 18) was explored by
studying all except 2 cases in the series for the presence of
either LMP-I protein or EBER-l transcript. EBV was detectable in 6 cases, only l ofwhich showed strong expression
of hcl-2. This case, in which there was no history of follicular
lymphoma, was among the4 cases with strong bel-2 expression and nodetectable t( 14; 18) or history of follicular lymphoma. Therefore, we were unable to find a plausible explanation for the enhanced hcl-2 expression in 3 of the cases in
our primary series. Although we cannot rule out thepossible
occurrence oft( 14; 18)sthat involve breakpoints outside of
the MBR or MCR. which our PCR primers would not have
detected, it is also possible that elevated hcl-2 expression in
H D cases may be brought about by other molecular mechanisms, including inductionby viruses other thanEBV.
Others have studied the t( 14: 18) in H D by PCR. Some
investigators have found a low but significant prevalence.
with the highest being 17 of 53 cases (32%).6-9However, several groups have been unable to identify any cases with the
t r a n s l ~ c a t i o n . ~Poppema
~ - ~ ~ et aIs5detected the t( 14; 18) in
1 1 of 28 cases by PCR but in only1 of these cases by cytogenetics and suggested that in some HD tissues the translocation might be located in “background” lymphocytes rather
than Hodgkin cells.55This would be consistent with the reported finding of t(14: 18) in benign, hyperplastic lymphoid
t i s s ~ e s . ’ ~Reviews
. ~ ~ ofcytogenetic studiesalso support a low
incidence oft( 14; 18) in HD.58.59Although abnormalities of
band 14q32 have been reported in a number of fresh tissue
specimens as well as in cell lines, thet(14;18)(q32;q21)
translocation
has
been specifically recognized only
rareI~.~~.~O.~’
The discrepancies in PCR results are difficult to explain,
but at least some may be related to technical differences.
Unlike several other groups, we used control samples containing approximately
pg of DNA (roughly the amount
present in 10 human cells and far fewer than theanticipated
number of Hodgkin cells in any of the samples) from lymphomas known to carry a 14; 18 translocation to define the
lower limit of hybridization signal intensity that we would
consider as a “positive” result.
If follicular lymphoma does, in fact, occasionally progress
to HD, it is difficult to estimate the relative contribution of
such a process to cases of HD occurring inthe general population. A prior history of follicular lymphoma is rare
among patients with HD, although probably not so rare as
From www.bloodjournal.org by guest on November 17, 2014. For personal use only.
228
LEBRUN ET AL
Table 5 . HD Associated With Follicular Lymphoma
Follicular Lymphoma
Patlent
lnltials
FLC
HH
970) (1
HN
vs
88
FSC
HE
MH
AL
KM
FSC
HR
FSC
LD
BR
HD
(year of dlagnosls)
(year of dlagnosls)
985)
NSHD (1
FSC(1976)
983) FSC (1
983) FSC (1
989)
MCHD ( 1 987)
992)
Yes
++
CD1
++
Yes
++
CD1
No
ND
989)
NSHD (1
++
CD1
ND
(1
HD ( 1 987)
MCHD (1 983)
NSHD (1 988)
(1
990)
MCHD (1
(1
t(14:181
++
987) NSHD (1
990)
MCHD (1
FLC(1987)
977)
FSC (1
FSC (1988)
990)
b d 2 Staining In
Hodgkln Cells
993) HD (1
986)F&DLC (1
986)MCHD (1
++
++
++
ND
ND
ND
++
CD1
ND
++
CD1
ND
?
CD1
ND
Hodgkln Cell Phenotype’
CD15+ CD30’ CD20
CD45R- CD5- CD4- CD8
5+ CD30’
CD45RB- CD43- CD37
CD1 5+CD45RB5+ CD30~CD45RB
CD20- CD45RO- CD43
5+ CD45RB- CD20
CD43CD 15+CD45R8
CD1 5’ CD30’ C D 2 0 ~ ~
CD15- CD20 CD43
CD45RB5’ CD30- CD20CD43 - CD45RB5’ CD30+ CD20~
CD37- CD435+CD45RB
Abbreviations: FLC, follicular large cell; FSC, follicular small cleaved cell; F&DLC, follicular and diffuse large cell; NSHD, nodular sclerosing HD; MCHD,
strong staining; ?, result uninterpretable due to absence of stainmg in background lymphocytes; ND not determined.
mixed cellularity HD;
* Phenotyping was performed on frozen or paraffin-embedded tissue sections.
++,
in the general population. In a study published in 1948, Custer and Bernhard62reviewed 700 cases of HD andidentified
4 patients (0.57%) in whom follicular lymphoma had preceded HD. Interestingly, no cases were found in which H D
had preceded follicular NHL. The relatively large proportion of HD patients in our initial series who had previously
been treated for follicular lymphoma (almost 10%)almost
certainly represents an overestimate and may be related to
the large number of patients with follicular lymphoma who
receive long-term follow-up at our institution. A high prevalence of these patients inour series relative to thatin other
studies might account for the failure of previous studies to
detect an association between t( 14; 18) in HD and a prior
history of follicular lymphoma.
ACKNOWLEDGMENT
The authors gratefully acknowledge the technical assistance of
Carmencita Nicholas and Eva Pfendt and the photographic assistance of Philip Verzola. We are also grateful to Dr D.Y. Mason
(Oxford University, Oxford, UK) and DrA.B. Rickinson (University of Birmingham, Birmingham, UK) for providing MoAbs for
bcl-2 and LMP-1, respectively.
REFERENCES
I . Sundeen J, Lipford E, Uppenkamp M, Sussman E, Wahl L,
Faffeld M, Cossman J: Rearranged antigen-receptor genes in Hodgkin’s disease. Blood 70:96, 1987
2. Weiss LM, Strickler JG, HuE, Warnke RA, Sklar J: Immunoglobulin gene rearrangements in Hodgkin’s disease. Hum Pathol
17:1009, 1986
3. Schmid C, Pan L, Diss T, lsaacson P: Expression of B-cell antigens by Hodgkin’s and Reed-Sternberg cells. Am J Pathol 139:
701, 1991
4. Jaffe ES: The elusive Reed-Sternberg cell. N Engl J Med 320:
529, 1989
5. Yunis JJ, Oken MM, Kaplan ME, Ensrud KM, Howe RR.
Theologides A: Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin’s lymphomas. N Engl J Med 307:
1231, 1982
6. Stetler-Stevenson M, Crush-Stanton S, Cossman J: Involvement of the hcl-2 gene in Hodgkin’s disease. J Natl Cancer Inst 82:
855. 1990
7. Reid AH, Cunningham RE, Frizzera G, O’Leary TJ: BCL-2
rearrangement in Hodgkin’s disease. Results of polymerase chain
reaction, flow cytometry and sequencing on formalin-fixed, paraffin-embedded tissue. Am J Pathol 142:395, 1993
8. Gupta RK. Whelan JS. Lister TA, Young BD, Bodmer JG:
Direct sequence analysis of the t( 14; 18) chromosomal translocation
in Hodgkin’s disease. Blood 79:2084, 1992
9. Lorenzen J, Hansmann M, Pezzella F, Hesse C, Kneba M.
Gatter KC, Fischer R: Expression of the bcl-2 oncogene product
and chromosome translocation t( 14: 18) in Hodgkin’s disease. Hum
Pathol23: 1205, 1992
10. Chen-Levy Z, Nourse J, Cleary M: The bcl-2 candidate protooncogene product is a 24-kilodalton integral membrane protein
highly expressed in lymphoid cell lines and lymphomas carrying the
t(14; 18) translocation. Mol Cell Biol9:701, 1989
I I . Vaux DL, Cory S, Adams JM: Bcl-2 gene promotes hematopoietic cell survival and cooperates with c-myc to immortalize B
cells. Nature 335:440, 1988
12. Hockenbery DM, Nunez G, Milliman C, Schrieber RD,
Korsmeyer SJ: Bcl-2 is an inner mitochondrial membrane protein
that blocks programmed cell death. Nature 348:334, 1990
13. Nunez G, London L, Hockenbery
D,
Alexander M,
McKearn JP, Korsmeyer SJ: Deregulated bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic
cell lines. J Immunol 144:3602, 1990
14. Henderson S, Rowe M, Gregory C, Croom-Carter D. Wang
F, Longnecker R, Kieff E, Rickinson A: Induction of bcl-2 expres-
From www.bloodjournal.org by guest on November 17, 2014. For personal use only.
bcl-2 ANDHODGKIN’SDISEASE
sion by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell 65: I 107, 1991
15. Lukes RJ: Criteria for involvement of lymph nodes, bone
marrow, spleen, and liver in Hodgkin’s disease. Cancer Res 31:
1755, 1971
16. Doggett RS, Colby TV, Dorfman R F Interfollicular Hodgkin’s disease. Am J Surg Pathol7: 145, 1983
17. Cleary ML, Chao J, Warnke RA, Sklar J: Immunoglobulin
gene rearrangement as a diagnostic criterion of B-cell lymphoma.
Proc Natl Acad Sci USA8 1593, 1984
18. Cleary ML, Smith SD,Sklar J: Cloning and structural analysis of cDNA’s for bcl-2 and a hybrid bc/-2/imrnunoglobulin transcript resulting from the t(14; 18) translocation. Cell 47: 19,1986
19. Ngan B-Y, Nourse J, Cleary M L Detection ofchromosomal
translocation t( 14; 18) within the minor cluster region of bcl-2 by
polymerase chain reaction and direct genomic sequencing of the
enzymatically amplified DNA in follicular lymphomas. Blood 73:
1759, 1989
20. Cleary ML, Galili N, Sklar J: Detection of a second t( 14; 18)
breakpoint cluster region in human follicular lymphomas. J Exp
Med 164:315, 1986
2 I . Pezzella F, Tse A, Cordell JL, Pulford KAF, Gatter KC, Mason DY: Expression of the bcl-2 oncogene proteinis not specific for
the 14; I8 chromosomal translocation. Am J Pathol 137:225, 1990
22. Bindl JM, Warnke RA: Advantages ofdetecting monoclonal
antibody binding to tissue sections with biotin and avidin reagents
in Coplin jars. Am J Clin Pathol85:490, 1986
23. Rowe M, Evans HS, Young LS, Hennesy K, l e f f E, Rickinson AB: Monoclonal antibodies to the latent membrane protein
and inducible expression in virus-transformed cells. J Gen Virol68:
1575, 1987
24. Weiss LM, Chen Y-Y, Liu X-F, Shibata D: Epstein-Barr virus and Hodgkin’s disease: A correlative in situ hybridization and
polymerase chain reaction study. Am J Pathol 139:1259, 199 I
25. Saiki RK, Gelfand DH, Stoffel S, ScharkSJ, Higuchi R,
Horn GT, Mullis KB, Erlich HA: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science
239:487, 1988
26. Cunningham D, Hickish T, Rosin RD, Sauven P, Baron JH,
Farrell PJ, Isaacson P: Polymerase chain reaction for detection of
dissemination in gastric lymphoma. Lancet 1:695, 1989
27. Lee M-S,Chang K-S, Cabanillas F, Freireich EJ, Trujillo JM,
Stass SA: Detection of minimal residual cells carrying the t( 14; 18)
by DNA sequence amplification. Science 237: 175, 1987
28. Crescenzi M, Set0 M, Herzig GP, Weiss PD, Griffith RC,
Korsmeyer SJ: Thermostable DNApolymerase chain amplification
oft( 14; 18) chromosome breakpoints and detection of minimal residual disease. Proc Natl Acad Sci USA 85:4869, 1988
29. Stetler-Stevenson M, Raffeld M, Cohen P, Cossman J: Detection of occult follicular lymphoma by specific DNA amplification. Blood 72:1822, 1988
30. Kim H, Hendrickson R, Dorfman RF: Composite lymphoma. Cancer 40:959, 1977
3 I . Hansmann ML, Fellbaum C, Huis PK,Lennert K: Morphological and immunohistochemical investigation of non-Hodgkin’s
lymphoma combined with Hodgkin’s disease. Histopathology 15:
35, 1989
32. Gonzales CL. Medeiros J, Jaffe ES: Composite lymphoma.
A clinicopathologic analysis of nine patients
with Hodgkin’s disease
and B-cell non-Hodgkin’s lymphoma. Am J Clin Pathol 96:81,
1991
33. Coltman CA, Dixon DO: Second malignancies complicating
Hodgkin’s disease. A Southwest Oncology Group 10 year follow up.
Cancer Treat Res 66: 1023, 1982
229
34. Armitage JO, Dick FR, Goeken JA, Foucar K,Gingrich RD:
Secondlymphoidmalignantneoplasmsoccurring
in patients
treated for Hodgkin’s disease. Arch Intern Med 143:445, 1983
35. Zarate-Osorno A, Medeiros J, Kingma D, Longo D, Jaffe E:
Hodgkin’s disease following non-Hodgkin’s lymphoma. A clinicopathologic and immunophenotypicstudy of nine cases. Am J Surg
Pathol 17:123, 1993
36. Stein H, Mason DY, Gerdes J, OConnor N, Wainscoat J,
Pallesen G, Catter K, Falini B, Delsol G, Lemke H, Schwarting R,
Lennert K: The expression of the Hodgkin’s disease-associated antigen Ki- 1 in reactive and neoplastic lymphoid tissue: Evidence that
Reed-Sternberg and histiocytic malignancies are derived from activated lymphoid cells. Blood 66:848, 1985
37. DeJongD, Voetdijk BMH, Beverstock GC, van Ommen
GJB, Willenze R, Kluin PM: Activation of the c-myc oncogene in a
precursor-B-cell blast crisis presenting as composite lymphoma.N
Engl J Med 318:1373, 1989
38. McDonnell TJ, Korsmeyer SJ: Progression from lymphoid
hyperplasia to high-grade malignant lymphoma in mice transgenic
for the t( 14; 18). Nature 349:254, I99 I
39. Brecher M, Banks P Hodgkin’s disease variant of Richter’s
syndrome. Report ofeight cases. Am J Clin Pathol93:333, 1990
40. Williams J, Schned A, Cotelingam JD, Jaffe ES: Chronic
lymphocytic leukemia with coexistant Hodgkin’s disease. Implications for the origin of the Reed-Sternberg cell. Am J Surg Pathol 15:
33, I99 I
4 I . Chan WC, Melvin CL, Grozea PN, Free1 R J , Variakojis D:
Mycosis fungoides and Hodgkin’s disease occurring in the same
patients: Report of three cases. Cancer 44: 1408, l979
42. Donald D, Green J, White M: Mycosis fungoides associated
with nodular sclerosing Hodgkin’s disease. Cancer 46:2505, 1980
43. Gregory CD, Dive C, Henderson S, Smith CA, Williams GT,
Gordon J, Rickinson AB: Activation of Epstein-Barr virus latent
genes protects human B cells from death by apoptosis. Nature 349:
612, 1991
44. Anagnostopoulos I, Herbst H, Niedobitek G, Stein H: Demonstration of monoclonal EBV genomes in Hodgkin’s disease and
ki- 1 positive anaplastic large cell lymphoma by combined Southern
blot and in situ hybridization. Blood 7 4 3 IO, 1989
45. Weiss LM, Movahed LA, Warnke RA, Sklar J: Detection of
Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin’s
disease. N Engl J Med 320502, 1989
46. Herbst H, Dallenbach F, Hummel M, Niedobitek G, Pileri
S, Muller-Lantzsch N, Stein H: Epstein-Barr virus latent membrane
protein expression in Hodgkin and Reed-Stemberg cells. Proc Natl
Acad Sci USA 88:4766, 199 I
47. Brousset P, Chittal S, Schlaifer D, kart J, Payen C, RigalHuguet F, Voigt J-J, Delsol G: Detection of Epstein-Barr virus messenger RNA in Reed-Stemberg cells of Hodgkin’s disease by in situ
hybridization with biotinylated probes on specially processed modified acetone methyl benzoate xylene (ModAMeX) sections. Blood
77:1781, 1991
48. Pallesen G, Hamilton-Dutoit SJ, Row M, Young LS: Expression of Epstein-Barr virus latent membrane gene products in tumour cells of Hodgkin’s disease. Lancet 337:320, 1991
49. Algara P, Martinez P, Sanchez L, Villuendas R, Orradre JL,
Oliva H, Pins MA: Lymphocyte predominance Hodgkin’s disease
(nodular paragranuloma)-A bcl-2 negative germinal centre lymphoma. Histopathology 19:69, 1991
50. Louie DC, Kant JA, Brooks JJ, Reed JC: Absence oft( 14; 18)
major and minor breakpoints and of bcl-2 protein overproduction
in Reed-Sternberg cells of Hodgkin’s disease. Am J Pathol 139:
1231, 1991
From www.bloodjournal.org by guest on November 17, 2014. For personal use only.
230
5 I . Said JW, Sassoon AF. Shintaku IP, Kuritin PJ. Pinkus CS:
Absence of hcl-2 majorbreakpoint
region andJH
gene rearrangement in lymphocyte predominance Hodgkin’s disease. Results of Southern blot analysis and polymerase chain reaction. Am
J Pathol 138:26I , l99 1
52. Shibata D. Hu E, Weiss LM. Brynes RK, Nathwani BN: Detection of specific t( 14: 18) chromosomal translocations in fixed tissues. Hum Pathol21 : 199. I990
53. Segal CH, Wittwer CT. Fishleder AJ, Stoler MH. Tubbs RR.
Kjeldsberg CR: Identification of monoclonal B-cell populations by
rapid cycle polymerase chain reaction. A practical screening
method for the detection of immunoglobulin gene rearrangements.
Am J Pathol 141:1291. 1992
54. Athan E, Chadburn A. Knowles D: The BCL-2 gene translocation is undetectable in Hodgkin’s disease by Southern blot hybridization and polymerase chain reaction. Am J Pathol 141: 193.
I992
55. Poppema S, Kaleta J. Hepperle B: Chromosomal abnormalities in patients with Hodgkin’s disease: Evidence for frequent involvement of the 14q chromosomal region but infrequent BCL-2
gene rearrangement in Reed-Sternberg cells. J Natl Cancer lnst 84:
1789. I992
LEBRUN ET AL
56. Limpens J. de Jong D. van Kriekin J. Price C. Young B, van
Ommen G-J, Kluin P: BCL-2/JH rearrangements in benign lymphoid tissues with follicular hyperplasia. Oncogene 6227 I . 199 I
57. Aster J. Kobayashi Y, Shiota M, Sklar J: Detection of the
t( 14: 18) at similar frequencies in hyperplastic lymphoid tissues
from American and Japanese patients. Am J Pathol 14 I :29 1. 1992
58. Cabanillas F: A review and interpretation ofcytogenetic abnormalities identified in Hodgkin’s disease. Hematol Oncol 6:27 I .
I988
59. Thangevelu M. LeBeau MM: Chromosomal abnormalities
in Hodgkin’s disease. Hematol Oncol CIin North Am 3 2 2 I . 1984,
60. Naumovski L, Utz PJ. Bergstrom SK. Morgan R. Molina A.
Toole JJ. Glader BE, McFall P. Weiss LM. Warnke R. Smith SD:
SUP-HDI: A new Hodgkin’s disease-derived cell line with lymphoid features produces interferon-?. Blood 74:2733. 1989
6 I . Cabanillas F. Pathak S. Trujillo J. Grant G. Cork A. Hagemeister FB, Velasquer WS. McLaughlin P, Redman J. Katr A: Cytogenetic features of Hodgkin’s disease suggest possible origin from
a lymphocyte. Blood 7 I : 16 15. l988
62. Custer RP, Bernhard WG: The interrelationship of Hodgkin’s disease and other lymphatic tumors. Am J Med Sci 216:625.
I948