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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 1023-1031
NEOPLASIA
Hodgkin and Reed-Sternberg-like cells in B-cell chronic
lymphocytic leukemia represent the outgrowth of single germinal-center
B-cell-derived clones: potential precursors of Hodgkin and
Reed-Sternberg cells in Hodgkin's disease
Holger Kanzler,
Ralf Küppers,
Sabine Helmes,
Hans-Heinrich Wacker,
Andreas Chott,
Martin-Leo Hansmann, and
Klaus Rajewsky
From the Institute for Genetics and the Departments of Internal
Medicine and Pathology, University of Cologne, Cologne, Germany; the
Department of Hematopathology, University of Kiel, Kiel, Germany; the
Department of Clinical Pathology, Allgemeines Krankenhaus, Vienna,
Austria, and the Institute for Pathology, University of Frankfurt,
Frankfurt, Germany.
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Abstract |
In rare cases of B-cell chronic lymphocytic leukemia (B-CLL), large
cells morphologically similar to or indistinguishable from
Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin's disease (HD) can be
found in a background of otherwise typical B-CLL. To test these
HRS-like cells for a potential clonal relationship to the B-CLL cells,
single cells were micromanipulated from immunostained tissue sections,
and rearranged immunoglobulin genes were amplified from
HRS-like cells and B-CLL cells and sequenced. The same variable (V) gene rearrangements with shared and distinct
somatic mutations were found in HRS-like and B-CLL cells from 1 patient, which indicates derivation of these cells from 2 distinct
members of a germinal-center B-cell clone. Separate clonal V
gene rearrangements were amplified from HRS-like and B-CLL cells from 2 other patients, showing concomitant presence of 2 distinct expanded
B-cell clones. Epstein-Barr virus (EBV) was detected in the HRS-like
cells of these 2 latter cases, indicating clonal expansion of an
EBV-harboring B cell in the setting of B-CLL. There is
evidence that HRS-like cells in B-CLL, like HRS cells in HD, derive
from germinal-center B cells. In all cases, somatic mutations have been
detected in the rearranged V genes of the HRS-like cells, and
in 1 of the EBV-positive HRS-like cell clones, somatic mutations
rendered an originally functional V gene rearrangement
nonfunctional. We speculate that the HRS-like cells in B-CLL represent
potential precursors for HRS cells causing HD.
(Blood. 2000;95:1023-1031)
© 2000 by The American Society of Hematology.
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Introduction |
Hodgkin and Reed/Sternberg (HRS) cells
represent a histological hallmark and, if surrounded by a
typical mixed cellular infiltrate, are indicative of the diagnosis of
Hodgkin's disease (HD). The occurrence of cells with the morphology
and often the immunophenotype of HRS cells (eg, positivity for the
surface antigens CD30 and CD15) has been described in rare cases of
B-cell chronic lymphocytic leukemia (B-CLL).1,2 In some
instances, the HRS-like cells reside scattered in a background of CLL
in the absence of the rich cellular infiltrate of HD.2,3
Often Epstein-Barr virus (EBV) is present in the HRS-like cells,
suggesting that EBV infection may play a role in the generation of
these cells.3,4 Since it is now known that the HRS cells in
HD are usually derived from germinal-center (GC) B cells,5
it is an intriguing question whether also the HRS-like cells in
diseases other than HD are B-lineage derived and related to GC B cells.
In approximately half of the cases,3 a
B-lineage origin of the HRS-like cells in B-CLL is indicated by the
expression of B-cell-specific antigens. Furthermore, it has long been
unclear whether the HRS-like cells in B-CLL are clonally related to the
underlying CLL and perhaps represent phenotypic variants of the
original lymphoma. A recent study by Ohno et
al6 described rearranged immunoglobulin V region genes
(IgV) in HRS-like and B-CLL cells in Richter's syndrome, which
represents a blastic transformation of a B-CLL. Three cases studied
indeed showed that the HRS-like cells can represent B cells clonally
related to the concurrent B-CLL cells because identical V gene
rearrangements were amplified from those cells in 2 of the cases.
We applied polymerase chain reaction (PCR) analysis of single
micromanipulated cells for IgV gene rearrangements7
to further study the clonal relationship of HRS-like cells and
concurrent B-CLL cells and to investigate the differentiation stage of
the HRS progenitors, which was not addressed by Ohno et
al.6
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Materials and methods |
Patients and tissues
Patient 1 was an 85-year-old female with a previous history of CLL;
the white blood cell count was 19 × 109/L at
diagnosis of CLL in November 1988. Adenocarcinoma was diagnosed and BII
gastrectomy performed. No further therapy, such as radiotherapy or
chemotherapy, was given. Biopsy of a supraclavicular lymph node in
January 1995 showed an infiltration of B-CLL with Hodgkin-like cells
and a small number of Reed-Sternberg-like cells interspersed in the
CLL tumor B cells. The white blood cell count in January 1995 was
32 × 109/L. No further biopsies were performed, the
CLL continued, and the patient died in April 1995.
Patient 2 was a 61-year-old female who presented in May 1981 with an
axillary lymph node infiltrated by CLL and a high proliferation activity of the tumor. In December 1986, the patient presented with
enlarged lymph nodes at different locations. The white blood cell count
was 42 × 109/L. Chemotherapy was performed
according to the COP regimen (cyclophosphamide, vincristine
[Oncovin], and prednisone). The histology of a lymph node biopsy
taken in January 1987 revealed B-CLL with intermingled HRS-like cells.
HRS-like and B-CLL cells were isolated from this biopsy specimen. The
white blood cell count in February 1987 was 17 × 109/L. The patient received 6 cycles of
chemotherapy according to the COP-ABV-IMEP regimen, which comprises
COP-doxorubicin (Adriamycin), bleomycin, and vinblastine-ifosfamide,
methotrexate, etoposide, and prednisone. Lymph node
histology in November 1987 revealed infiltration by CLL with no
indication of HRS-like cells or HD. The white blood cell count was
42 × 109/L. In April 1988 the patient presented
again with enlarged lymph nodes; the lymphoma had progressed, and the
white blood cell count was 84 × 109/L. The patient
received 1 cycle of chemotherapy according to the CHOP-regimen
(cyclophosphamide, doxorubicin, vincristine [Oncovin], prednisone). We did not follow this patient further because she did not
return to follow-up.
Patient 3 was admitted to the hospital in September 1997 at the age of
47 because of left-sided axillary lymphadenopathy. The patient's
clinical history did not reveal preceding neoplastic or autoimmune
disorders; however, the diagnosis of infectious mononucleosis was made
at an outside hospital in 1995. An axillary lymph node biopsy was
taken, resulting in a diagnosis of B-CLL with scattered HRS-like cells.
The white blood cell count was 8.0 × 109/L with
51% lymphocytes and no eosinophilia or monocytosis. Flow cytometry
immunophenotyping of peripheral blood and bone marrow revealed the
presence of an abnormal
CD19+CD5+CD23+ lymphoid cell
population, and bone marrow trephine showed 60% diffuse infiltration
with small lymphocytes consistent with B-CLL. Atypical large cells with
or without features of HRS cells were not present. The patient was
treated with local radiation therapy (total 36 Gy) to the left axilla
involving the adjacent supraclavicular and cervical regions in addition
to 6 cycles of combination chemotherapy according to the CHOP regimen.
Complete clinical and hematological remission was achieved in March
1998. Restaging in August 1998 and abdominal computed tomography (CT)
scans revealed a solitary lesion localized between the aorta and
inferior vena cava; it was not biopsied. In September 1998 the patient
presented with paraaortal lymphadenopathy, and an autologous peripheral
blood stem cell transplantation was performed in January 1999. Staging at day 100 by thoracic and abdominal CT scans was negative; however, bone marrow phenotyping revealed 41% CD5+CD19+
mononuclear cells, which indicated persistent marrow involvement by
B-CLL.
Immunostaining and in-situ hybridization
Immunological studies were performed on frozen and/or
paraffin-embedded tissues. Sections were stained with antibodies
against CD30, CD20, and LMP1 (BerH2, L26, and CS1-4, respectively;
Dako, Hamburg, Germany); CD15 and CD5 (LeuM1 and DK23, respectively; Becton Dickinson, Mountain View, CA); and CD3 (OKT3; Ortho Diagnostic Systems, Raritan, NJ) using the avidin-biotin-complex technique (Dako)
as described.8 Fast red or new fuchsin was used as
substrate for the alkaline phosphatase (AP).
In-situ hybridization (ISH) for detection of EBV-encoded small nuclear
RNAs (EBER1 and 2) was performed using in vitro transcribed digoxygenin-labeled sense and antisense EBER probes.9 In
brief, dewaxed and rehydrated tissue sections were treated with
hydrochloride (0.1 N) and Pronase (1.25 µg/mL)
(Boehringer Mannheim, Mannheim, Germany). Sections were refixed in 4%
paraformaldehyde, dehydrated through graded ethanols, and hybridized
for 18 hours at 37°C. The excess on the probe was removed by
washing it with 2 × SSC (standard saline citrate)/50% formamid
and 0.1 × SSC/50% formamid, followed by digestion with 20 µg/mL ribonuclease for 20 minutes at 37°C. Slides were
washed with 2 × SSC and 0.1 × SSC. Bound digoxygenin-labeled probes were detected by digoxygenin-AP-coupled Fab
fragment, and bound AP was visualized by fast red substrate.
Isolation of DNA from tissue sections
DNA was extracted from frozen or paraffin-embedded tissue sections
by standard methods10 or by using a tissue kit (QiaAmp Tissue kit; Qiagen, Hilden, Germany).
Micromanipulation of cells
Immunostained frozen sections (6-µm to 8-µm thick) were overlaid
with tris(hydroxymethyl) aminomethane-buffered
(Tris-buffered) saline, and single cells were mobilized with the help
of hydraulic micromanipulators (Narishige, Tokyo, Japan)
on an inverted microscope (Olympus, Hamburg, Germany).8
Cells were transferred into 20 µL PCR buffer supplemented with 1 ng/µL 5S ribosomal RNA (rRNA) (Boehringer Mannheim) and
stored at  80°C. Single CD30-positive HRS-like cells and single
CD3-positive T cells as well as CD20-positive B cells of the CLL were
obtained from adjacent sections. In some micromanipulation experiments,
the B cells of the CLL were identified as small CD3-negative or
CD30-negative cells. Samples of the buffer covering the sections were
aspirated, and they served as negative controls for the PCR
amplification. In the repeat experiments, separate sections were
analyzed following the procedure described above.
Preamplification of genomic sequences
In some experiments, the genomic DNA of single cells was amplified
following the primer-extension preamplification (PEP) approach of Zhang
et al,11 using proteinase K incubation (2 hours at 50°C
with 0.3 mg/mL proteinase K [PCR grade, Boehringer Mannheim]) instead
of alkali denaturation to isolate DNA. From these reactions, 4 µL
aliquots were analyzed for rearranged IgV genes, as described below.
PCR analysis
Rearranged VH,
V , and V genes were
amplified in a seminested PCR using family-specific V gene primers
together with 2 sets of the respective joining (J) gene segment primers
as described.8,12-14 The PCR conditions are summarized in
Table 1. The sequences of the
V3 gene family-specific leader primers
are: V3L1 5' CAC CAT GGC CTG GAC CCC TCT CTG
3', V3L2 5' CAC CAT GGC CTG GAY CCC TCT MCT
3', V3L3 3' ATG GCA TGG ATC CCT CTC TTC CTC
G 3', V3L4 5' GCC ATG GCC TGG ACC GYT CTC CT
3'.
Rearranged IgV genes from DNA extracted from fresh tissue
sections were amplified as described for the second round of PCR using
35 cycles of amplification. PCR products were gel-purified and directly
sequenced. For sequencing of PCR products obtained from B-CLL cells of
patient 3, primers binding to the complimentarity determining region II
(CDRII) of the in-frame VH3 gene
rearrangement (IF 5' ATT AGT GGT AGT GGT GGT AG 3') or to
the CDRII of the out-of-frame VH3 gene
rearrangement (OOF 5' GCT ATT GGT ACT GCT GGT GA 3') were
used. This was done because the 2 VH3 gene
rearrangements were repeatedly coamplified from the same samples, which
resulted in mixed sequences when the VH3 FRI primer was
used for sequencing. Sequences were analyzed using software (DNASIS;
Pharmacia Biotech, Uppsala, Sweden); a genetic library
(GenBank library, http://www.ncbi.nlm.nih.gov/BLAST); and
a database (IMGT;
http://www.genetik.uni-koeln.de/dnaplot/).
| |
Results |
Histology and immunohistology of B-CLL-affected lymph nodes
Lymph node biopsies from 3 patients with B-CLL were investigated,
and histological sections of all cases showed features consistent with
B-CLL. The typical polymorphic lympho-histiocytic cellular infiltrate
of HD was absent in all cases.
In case 1, the supraclavicular lymph node was infiltrated with small
lymphocytes (Figure 1A). Some histiocytes
with broad cytoplasm were intermingled. Medium- to large-size cells
showing features of Hodgkin cells were randomly admixed, and
binucleated cells resembling Reed-Sternberg cells were low in number.
In case 2, the lymph node showed an infiltrate consisting of
lymphocytes with round to slightly irregularly shaped nuclei and
histiocytes (some with epitheloid differentiation). Hodgkin-like and
numerous Reed-Sternberg-like cells were solitarily intermingled
(Figure 1B-D). Occasionally small- to medium-size proliferation centers could be identified. In case 3, the lymph node structure was dominated by small lymphocytes showing some centers of proliferation. In addition
to HRS-like cells, Hodgkin, classical Reed-Sternberg, and
"mummified" cells were seen occasionally (Figure 1E). Many blood
vessels were found, and histiocytes occasionally showed an epitheloid
differentiation. In all 3 cases a slight to moderate positive
immunostaining for the  light chain and IgM was found in small- to
medium-size lymphocytes (not shown). In the proliferation centers of
all cases, some of the blast cells showed a perinuclear positivity for
immunoglobulin. HRS-like cells were either negative for Ig or could
show both light chains (likely reflecting unspecific adsorption of
serum Ig).
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| Fig 1.
Three cases of B-CLL with intermingled HRS-like cells.
(A, B, E) Anti-CD30 antibody staining of cases 1-3, hematoxylin
counterstain. Single CD30-positive HRS-like cells are interspersed in
the B-CLL infiltrated lymph node. (A) Case 1, 200 ×
magnification. (B) Case 2, 1000 × magnification. (E) Case 3, 400 × magnification. (C) Hematoxylin-eosin staining of case 2. The infiltrate mainly comprises small lymphocytes, a few blast cells,
and histiocytes. A Reed-Sternberg-like cell is seen in the middle of
the picture, 200 × magnification. (D) Hematoxylin-eosin staining
of case 2. A Reed-Sternberg cell surrounded by small lymphocytes and
histiocytes is seen in the middle of the picture, 1000 ×
magnification. (F) In situ hybridization of frozen section from
CLL-infiltrated lymph node of case 3 for EBER-RNA. The HRS-like cells
carry EBER-RNA, 400 × magnification. (A-F) Sections of
formalin-fixed, paraffin-embedded, or frozen tissue.
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Immunohistological studies showed that in all cases, the HRS-like cells
expressed the CD30 antigen, and in cases 1 and 3, they also expressed
CD15 (Table 2; Figure 1A, B, and E). The small lymphocytes of the CLL in all 3 patients were negative for CD30
and CD15; they were positive for the B-cell-associated CD20 antigen
and for CD5 (Table 2). EBV was detected in the HRS-like cells of
patients 2 and 3, but not in patient 1 (Table 2; Figure 1F). The small
CLL tumor cells were EBV-negative in all cases.
Micromanipulation and amplification of rearranged V
region genes
Single large HRS-like cells were isolated by micromanipulation from
CD30-stained frozen tissue sections of lymph node biopsies infiltrated
by B-CLL. B cells of the CLL were identified and isolated as either
CD20-positive, CD30-negative, or CD3-negative small cells. For patient
1, 3 micromanipulation experiments were performed, and 1 repeat
experiment was performed for patient 2 (Table
3). We used the following negative
controls: (1) single T cells obtained from CD3-stained adjacent
sections taken from the same tissue specimens on the same day as the
HRS-like and B-CLL cells and (2) aliquots of the buffer covering the
sections obtained during micromanipulation experiments. Genomic DNA of
some micromanipulated cells and controls was amplified following the
PEP protocol by Zhang et al.11 From aliquots of these
reactions, rearranged Ig genes were amplified. The
preamplification of genomic DNA was performed to investigate multiple
aliquots of the amplified DNA of a cell with different combinations of
primers and to preserve DNA for future analysis.
From each of the 3 cases analyzed, between 16 and 50 individual
HRS-like cells and at least 16 B-CLL cells were analyzed (Table 3). The
amplification efficiences for clonal Ig gene rearrangements ranged from 7% to 30% for the HRS-like cells (Table 3). The
corresponding efficiencies for the B-CLL cells could not be determined
because often 2 B-CLL cells were put into 1 reaction tube. These values were in the same range as efficiencies obtained in previous
micromanipulation studies.12,14,15 We believe that a given
rearrangement is not amplified from all cells largely due to technical
matters such as degradation or inaccessibility of the DNA. Moreover,
part of the nucleus is missing for many micromanipulated cells, and some rearrangements may not be efficiently amplified because of mutations at primer binding sites (see below).
In patient 1, HRS-like cells and B cells of the CLL harbor clonally
related V gene rearrangements with shared as well as distinct
somatic mutations
From patient 1, a total of 50 HRS-like cells were analyzed for
IgV gene rearrangements with 4 different combinations of
primers (Tables 1 and 3). The same potentially functional
VH4 gene rearrangement was amplified from
13 of 43 HRS-like cells analyzed for rearranged VH
genes. From 2 of the 28 cells analyzed for V
gene rearrangements, an identical potentially functional
V3 gene rearrangement was amplified, and
a nonfunctional V3 gene rearrangement was obtained from 5 cells. Sequencing of 25 out of 43 PCR products amplified from B-CLL cells revealed that the B-CLL cells carried the
same VH and V gene
rearrangements (Tables 3 and 4). Among the 25 sequenced PCR products,
the VH4 gene was obtained 15 times; the
potentially functional V3 gene 7 times;
and the nonfunctional V3 gene 3 times.
The VH and V gene
rearrangements amplified from HRS-like and B-CLL cells were somatically
mutated (Figure 2 and Table 4). No intraclonal sequence diversity
was observed among the clonally related V gene rearrangements
amplified repeatedly from HRS-like or B-CLL cells. The frequency of
mutations in the VH4 gene segment of the
HRS-like cells was 6.6%. In addition to the somatic mutations in the
VH4 gene of the HRS-like cells, 2 further point mutations were present in the B cells of the CLL (mutation frequency, 7.3%). The potentially functional
V3 gene rearrangement carried 11 identical somatic point mutations in the HRS-like and B-CLL cells
(Figure 2B) compared to the germline. One additional point mutation was
found in the HRS-like cells (mutation frequency, 5.3%), and 5 further
mutations were present in the respective gene rearrangement in the
B-CLL cells (mutation frequency, 6.7%). The nonproductively rearranged
V3 gene of the HRS-like and B-CLL cells
shared 25 mutations (sequences not shown). In the HRS-like cells, 1 additional point mutation was present, the gene rearrangement of the
B-CLL cells carried 6 further mutations, and an 8-base
pair (bp) duplication was found in framework region III (FRIII). The
presence of a high load of somatic mutations in the V gene
rearrangement amplified from the HRS-like and B-CLL cells may explain
the low efficiency of the amplification of the 2 V3 gene rearrangements from the cells.
Somatic mutations presumably also present at the primer binding sites
could severely reduce the amplification efficiency.
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| Fig 2.
V gene sequences from patient 1.
Sequences of (A) the VH4 gene rearrangement and (B)
the potentially functional V3 gene rearrangement
of the HRS-like and B cells of CLL are compared to the most homologous
V germline genes and J segments.35-38
Dashes indicate sequence identity. Codons are numbered according to
Kabat et al.34 CDRI-CDRIII are indicated.
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Table 4.
Sequence analysis of clonal Ig gene
rearrangements obtained from three cases of B-CLL with HRS-like
cells
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Further experiments were performed to amplify the region
spanning from the promoter to FRI of the potentially functional
VH4 gene rearrangement in overlap with the
sequences already generated. The purpose of this amplification was to
compare the HRS-like cells with the B-CLL cells and to investigate
whether somatic mutations may be present that would render this
potentially functional rearrangement nonfunctional. Such
"crippling" somatic mutations have been observed in HRS cells in
some cases of classical HD.12,15 However, the sequences of
the promoter and leader peptide region obtained were identical in
HRS-like and B-CLL cells, and mutations that would lead to a loss of
function of this gene rearrangement were not identified (sequences not shown).
From 1 of the 13 HRS-like cells and 1 of 25 T cells used as
negative controls, a VH4 gene rearrangement
was obtained whose sequences were identical to the corresponding
sequence of the B-CLL cells (Table 3). Most likely these sequences
result from cellular contamination by fragments of B cells of the CLL.
Since almost all cells in the tissue represent members of the B-CLL tumor clone, these cells represent a major risk of contamination. No
PCR product was amplified from a total of 13 buffer controls (Table 3).
The potentially functional VH4 and
V3 gene rearrangements obtained from
single B-CLL cells could also be amplified from genomic DNA extracted
from tissue sections of the investigated lymph node. This result shows
that the CLL tumor clone had been identified in the single-cell analysis.
The sequence of a second, slightly larger
V3 PCR product could not be determined
unambiguously, probably due to contamination of this amplificate with
the functional V3 gene rearrangement during isolation of the product from an agarose gel. This PCR product
most likely represents the second V3
gene rearrangement of the B-CLL tumor clone.
HRS-like cells in patients 2 and 3 harbor somatically mutated Ig
gene rearrangements that are clonal and not related to the V gene
rearrangements of the CLL clone
From patient 2, we investigated 33 HRS-like cells. An identical
VH3 gene rearrangement was amplified from
10 cells (Table 3). This rearrangement is somatically mutated,
with a mutation frequency of 8.2% (Figure
3; Table 4). This in-frame
VHDHJH gene rearrangement
had lost its functionality due to a stop codon in CDRII and a 1-bp
deletion in FRIII (Figure 3). A clonal nonfunctional V3 gene was amplified from 3 of 28 HRS-like cells investigated for V gene
rearrangements. This rearrangement has a mutation frequency of 9.5%
and shows a 13-bp deletion in FRI.
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| Fig 3.
"Crippling" somatic mutations in the
VH3 gene rearrangement from the HRS-like cells from
patient 2.
Comparison of the VH3 gene rearrangement from
HRS-like cells with the VH V3-30 germline
gene39 and the JH4
segment.38 ***Indicates a stop codon. A 1-bp deletion in
FRIII is indicated by  . CDRI -CDRIII are indicated.
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Of 36 samples containing 1 or 2 B cells of the CLL, 7 samples
gave rise to a total of 13 PCR products. The V gene
rearrangements of the B-CLL were not related to the rearrangements
obtained from the HRS-like cells (Table 3). A potentially functional
VH3 gene rearrangement was amplified 7 times from independent samples. Two samples gave rise to the same
potentially functional V3 gene
rearrangement. From 2 samples an identical nonfunctional V3 gene rearrangement was obtained and a
nonfunctional V2 gene rearrangement was
amplified once. The clonal VH3 and the V3 gene rearrangements and the
V2 region gene were also obtained from
genomic DNA extracted from the lymph node biopsy. (V gene rearrangements were not investigated.)
The clonal VH and VL
gene rearrangements of the B-CLL were unmutated (Table 4).
From 16 HRS-like cells of patient 3, an identical potentially
functional VH4 gene rearrangement was
amplified from 3 cells, and an identical nonfunctional
V1 gene rearrangement was amplified from
4 cells (Table 3). The VH4 and the
V1 gene rearrangements were somatically
mutated with mutation frequencies of 9.1% and 5.0%, respectively. A
1-bp deletion was present in FRII of the V1
gene.
V gene rearrangements amplified from the B-CLL cells were not related
to those of the HRS-like cells. From 16 samples containing 1 or 2 B-CLL
cells, an identical potentially functional
VH3 gene rearrangement was obtained from 4 samples; a nonfunctional VH3 gene
rearrangement was obtained from 5 samples; and an identical potentially
functional V1 gene rearrangement was
obtained from 4 samples. One of the 5 control T cells and 2 of the 10 buffer controls gave rise to the clonal V gene rearrangements
of the CLL, which was most likely due to cellular contamination. Clonal VH and VL gene
rearrangements of the B cells of the CLL were also obtained from
genomic DNA extracted from the lymph node biopsy. Except for a 1-bp
substitution in the nonfunctional VH3 gene
rearrangement, the V gene rearrangements of the B-CLL were unmutated (Table 4).
| |
Discussion |
Clonality and GC B-cell derivation of HRS-like cells in B-CLL
Clonal VH and VL gene
rearrangements were amplified from HRS-like cells of each of the 3 B-CLL cases. This shows that the HRS-like cells represent clonal
populations of mature B-lineage cells. These findings are in agreement
with a previous report by Ohno et al6 on expanded
populations of HRS-like cells with clonally related
VH gene rearrangements. In 2 of 3 cases of
Richter's syndrome with HD features, Ohno et al6 detected
identical VH gene rearrangements in single HRS-like
and B-CLL cells. In the third case, however, rearranged
VH genes were obtained from CLL cells but not from
HRS-like cells, and in that case, the relationship of the 2 cell
populations remained unexplained. Since only the CDRIII of IgH
gene rearrangements were amplified and sequenced, the presence of
somatic mutations remained uninvestigated, thereby preventing further
insight into the stage of differentiation of the HRS cell progenitor.
In the present study, sequence analysis of the rearranged V
genes of HRS-like cells revealed the presence of somatic mutations in
all rearrangements. The frequency of somatic mutation ranged from 5%
to 13.3% (Table 4). The detection of deletions or duplications in 4 of
the somatically mutated V region genes (3 V genes from HRS-like
cells and 1 V gene from B-CLL cells) is not unusual because such events have been repeatedly observed in normal and malignant human
B cells.16,17 Thus, in the 3 cases of B-CLL with HRS-like cells analyzed in the present work, the precursors of the HRS-like cells are likely to be GC B cells or descendants of these cells since
the process of somatic hypermutation is thought to be restricted to and
specific for human B cells undergoing proliferation and antigen
selection in GC.7
Studies on rearranged V genes in HRS cells in classical HD have
established that these cells in most cases represent clonal populations
of transformed GC B cells.5 In some of the cases of
classical HD, crippling somatic mutations were detected that rendered
originally productive V genes nonfunctional, thereby providing
evidence that HRS cells (or their precursors) are not selected for
antibody expression and have been rescued from apoptosis inside the GC
by some transforming event.5,12,15,18
In the present study, crippling somatic mutations were detected in the
originally potentially functional VH3 gene
of the HRS-like cells of patient 2 (Figure 3). However, in this case
(as opposed to several of the above-mentioned cases of HD with crippled
in-frame rearrangements) we cannot rule out the possibility that
another functional VH gene rearrangement was
present on the second IgH allele.14,19
Nevertheless, B cells carrying 2 productive VH region genes are rare, and we did not detect a second heavy chain gene
rearrangement. We consider it more likely that the HRS-like cells of
patient 2 carry a DJ gene rearrangement or
germline configuration in the second IgH allele and that the
HRS-like cells had lost the capacity to express an antigen receptor by
a crippling mutation. B cells acquiring crippling mutations during a GC
reaction are usually efficiently eliminated within the GC. It is thus
likely that the HRS-like cell clone in patient 2 is derived from a GC B
cell that was rescued from apoptosis by some transforming event such as
infection by EBV, which was detected in these cells. Thus, it appears
that also in diseases other than classical HD, cells phenotypically
resembling HRS cells can occur, which, like HRS cells of HD, derive
from crippled GC B cells.
B-CLL and HRS-like cells derived from a shared GC B-cell
precursor
In patient 1, the B-CLL cells carried somatically mutated clonal
VH and V gene rearrangements
(Tables 3 and 4). Approximately 50% of B-CLL cases carry somatically
mutated V genes.20 These cases are likely derived
from precursors that had passed through a GC reaction. Interestingly,
the vast majority of normal CD5-positive B cells carry unmutated
V region genes,21 which may indicate that CD5 B
cells driven into a GC reaction have an increased risk to undergo
malignant transformation.21
The HRS-like and B-CLL cells in patient 1 carried the same clonal
V gene rearrangements (Tables 3 and 4). Thus, the 2 cell populations turned out to be derived from a common precursor B cell.
Whereas most of the somatic mutations were shared between the HRS-like
and B-CLL cells, some mutations were present only in one or the other
of the cell types (Figure 2). This pattern of somatic mutation
indicates that a GC B cell which had already acquired somatic mutations
gave rise to 2 descendants, 1 of which developed to CLL, the other to
the HRS-like cell population (Figure 4).
Thus in this case, the pattern of somatic mutations also suggests a GC
B-cell derivation of the HRS-like cells. In addition, the mutation
pattern clearly argues against a disease scenario in which single
mature cells of the B-CLL clone occasionally and accidentally acquire
the phenotype of HRS-like cells. The derivation of the HRS-like and
B-CLL cells from a common precursor cell may indicate that distinct
molecular events caused the appearance of these 2 populations of
clonally expanded cells (Figure 4). In search for a transforming event
that could be involved in the generation of the 2 cell types, the cells
were analyzed for the presence of EBV. However, both cell types were
found to be EBV negative (Table 2).
View larger version (23K):
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| Fig 4.
Scenarios for the generation of B-CLL and associated
HRS-like cells.
Horizontal bars schematically depict rearranged V region genes, and
vertical bars depict somatic mutations. The shaded area represents a
germinal center.
|
|
The relationship of the B-CLL and the HRS-like cells in patient 1 is
reminiscent of 2 cases, representing combinations of HD and B
cell-non-Hodgkin's lymphoma (NHL) in the same patient, that we
recently analyzed.22 In both cases (one composite
follicular and Hodgkin's lymphoma, and a case of T-cell-rich B-cell
lymphoma in the skin followed by HD in a lymph node 3 years later), the HRS and B-NHL cells turned out to be clonally related and to represent distinct descendents of a shared GC B cell precursor.22
This was evident from the finding of shared as well as unique somatic mutations in the clonally related V genes of the 2 types of
tumor cells. Assuming that the HRS-like cells in patient 1 of the
present study represent malignant cells (a mere speculation at this
point), the HRS-like and B-CLL tumor cells would have likely undergone shared as well as distinct transforming events, which would allow the
appearance of 2 distinct populations of clonally related cells.
HRS-like cells in B-CLL: tumor precursors for HRS cells in HD?
Since the clonally related HRS-like and B-CLL cells in patient 1 likely share some transforming event (see above), one may speculate
that these HRS-like cells have an increased risk to develop to an
HRS-like cell clone of HD, perhaps upon acquiring additional
transforming events. In light of the increased risk of B-CLL patients
to develop HD as a secondary malignancy (see below), HRS-like cells
clonally related to the B-CLL, such as in patient 1, may represent
precursors for HRS tumor cells in HD following B-CLL.
In patients 2 and 3 a different scenario would have to be considered,
as in these cases the HRS-like cells were not related to the CLL tumor
clone (Tables 3 and 4, Figure 4). In patients 2 and 3, the HRS-like
cell clones were found to harbour EBV. As seen in EBV-positive
classical HD, the HRS-like cells analyzed here and observed previously
in cases of B-CLL3,4 express the EBV-encoded latent
membrane protein-1 (LMP-1), which is known for its B-cell transforming
capability.23 To explain the expansion of an EBV-harboring
B-cell clone in a setting of B-CLL, the following scenario can be
envisioned: In healthy EBV carriers, very rare B cells (1-50 per
106 cells) harbor EBV.24 These cells are
usually resting and express LMP-2 as the only EBV-encoded latent
antigen.25 However, the virus may sometimes change its
latency gene expression profile, thereby inducing proliferation of the
B cell.26 In healthy individuals, such proliferating
EBV-positive B cells are rapidly eliminated by an effective CD8 T-cell
response.27 However, B-CLL patients are often
immunosuppressed and have an impairment of cytotoxic T-cell
functions.28 Thus, in the setting of a B-CLL tumor,
reactivated EBV-positive B cells can perhaps clonally expand without
being eliminated by cytotoxic T cells. If this reactivation by
switching of the EBV gene expression profile is a rare event, the
proliferating EBV-positive B cells appearing in the patient at a given
point in time may often derive from a single precursor cell.
Alternatively, in the case of patient 3, the occurrence of HRS-like
cells may relate to the observation that this patient suffered from
infectious mononucleosis (the acute primary EBV infection) 2 years
before B-CLL was diagnosed. Since HRS-like cells infected by EBV
regularly develop during infectious mononucleosis,29,30 the
HRS-like cell clone in patient 3 might originate from an HRS-like cell
that was generated during the primary EBV infection and persisted in
the patient.
Interestingly, patients with B-CLL have approximately an 8-fold
increased risk to develop HD,31 with HD representing 1 of the most frequent secondary neoplasms (besides transformation of the
B-CLL into a large cell lymphoma, ie, Richter syndrome). In 4 patients
described in the literature, the EBV status of HRS-like cells of B-CLL
and the HRS cells of the concurrent or subsequent HD was studied. In
each instance, EBV was found in both types of HRS
cells.3,32 Thus, the HRS-like EBV-positive cell clones seen
in patients 2 and 3 may represent cells that sometimes, upon acquisition of additional transforming events (besides infection by
EBV), can develop to HD. That other events in addition to EBV infection
are needed to transform a B-CLL-associated HRS-like cell to an HRS
tumor clone causing HD is supported by the finding that the
lympho-histiocytic cellular infiltrate typical for HD is missing in
B-CLL with HRS-like cells. This cellular infiltrate is likely caused by
the HRS cells and is a major distinguishing feature of
HD.33
The present study provides evidence that HRS-like cells in B-CLL, like
HRS cells in HD, represent clonal populations of B lymphocytes derived
from GC B cells. These cells can either derive from the precursor that
also gave rise to the B-CLL or represent independent clones of
EBV-transformed B cells that expanded in the setting of a B-CLL. In
either case, HRS-like cells may potentially represent precursors for
HRS cells in HD.
| |
Acknowledgments |
We are grateful to A. Klöckner, A. Fassbender, and J. Jesdinsky
for excellent technical assistance and to T. Spieker for help
with the EBER in-situ hybridization. We thank A. Bräuninger for critical reading of the manuscript and R. Dalla-Favera for helpful discussion.
| |
Footnotes |
Submitted April 2, 1999; accepted September 29, 1999.
Supported by grants from Deutsche Forschungsgemeinschaft (SFB502) and
the Deutsche Krebshilfe, Dr Mildred Scheel Stiftung.
Reprints: Ralf Küppers, Department of Internal Medicine
and Institute for Genetics, LFI E4 R706, University of Cologne, Joseph-Stelzmannstr 9, 50931 Cologne, Germany.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction