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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2679-2687
Molecular Analysis of Single B Cells From T-Cell-Rich B-Cell
Lymphoma Shows the Derivation of the Tumor Cells From Mutating
Germinal Center B Cells and Exemplifies Means by Which
Immunoglobulin Genes Are Modified in Germinal Center B Cells
By
Andreas Bräuninger,
Ralf Küppers,
Tilmann Spieker,
Reiner Siebert,
John G. Strickler,
Brigitte Schlegelberger,
Klaus Rajewsky, and
Martin-Leo Hansmann
From the Department of Pathology, University of Frankfurt, Frankfurt;
the Institute for Genetics, University of Cologne, Cologne; the
Department of Human Genetics, University of Kiel, Kiel, Germany; and
the Department of Pathology, Mayo Clinic, Rochester, MN.
 |
ABSTRACT |
T-cell-rich B-cell lymphoma (TCRBCL) belongs to the group of
diffuse large cell lymphomas (DLL). It is characterized by a small
number of tumor B cells among a major population of nonmalignant polyclonal T cells. To identify the developmental stage of the tumor
progenitor cells, we micromanipulated the putative neoplastic large
CD20+ cells from TCRBCLs and amplified and sequenced
immunoglobulin (Ig) V gene rearrangements from individual cells. In six
cases, clonal Ig heavy, as well as light chain, gene rearrangements
were amplified from the isolated B cells. All six cases harbored
somatically mutated V gene rearrangements with an average mutation
frequency of 15.5% for heavy (VH) and 5.9% for light
(VL) chains and intraclonal diversity based on somatic
mutation. These findings identify germinal center (GC) B cells as the
precursors of the transformed B cells in TCRBCL. The study also
exemplifies various means how Ig gene rearrangements can be modified by
GC B cells or their malignant counterparts in TCRBCL: In one case, the
tumor precursor may have switched from  to  light chain
expression after acquiring a crippling mutation within the initially
functional light chain gene. In another case, the tumor cells
harbor two in-frame VH gene rearrangements, one of which
was rendered nonfunctional by somatic mutation. Either the tumor cell
precursor entered the GC with two potentially functional in-frame
rearrangements or the second VHDHJH
rearrangement occurred in the GC after the initial in-frame
rearrangement was inactivated by somatic mutation. Finally, in each of
the six cases, at least one cell contained two (or more) copies of a
clonal Ig gene rearrangement with sequence variations between these
copies. The presence of sequence variants for V region genes within
single B cells has so far not been observed in any other normal or
transformed B lymphocyte. Fluorescence in situ hybridization (FISH)
points to a generalized polyploidy of the tumor cells.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
IN THE REVISED European-American Lymphoma
(REAL) Classification, T-cell-rich B-cell lymphoma (TCRBCL) belongs to
the diffuse large cell lymphomas (DLL).1 TCRBCL is
characterized by few putative malignant B cells dispersed as single
cells in an infiltrate consisting mainly of T cells, histiocytes, and
plasma cells. The frequency of T cells can vary from 50% to more than 90% of cells in the tissue. The histology is usually diffuse, but some
cases may also contain nodular areas.2-4 The size of the
tumor B cells can be variable ranging from centroblast-like to
lymphocytic and histiocytic (L&H)-like cells, as they are found in
lymphocyte predominant Hodgkin's disease (LP HD).5 The
tumor cells are CD20+ and CD15- and
CD30-.6 In a few cases, they harbor the
Epstein-Barr virus (EBV).7,8 Besides the large neoplastic B
cells, varying amounts of small B lymphocytes (up to 50% of all cells)
are found in the tumor tissue.
TCRBCLs were first recognized as a separate entity by Ramsey et
al4 in 1988, demonstrating polyclonal T-cell receptor gene (TCR) rearrangements by Southern Blot analysis and clonal B-cell populations by monotypic immunoglobulin (Ig) light chain expression in
five lymphomas (previously diagnosed as peripheral T-cell lymphomas (PTCL)) with T-cell contents of more than 90%. By applying
immunohistochemistry and analysis of Ig and TCR gene rearrangements,
also other groups identified TCRBCLs among cases diagnosed previously
as PTCLs.6,7,9,10
Furthermore, the distinction of TCRBCL from the diffuse form of LP HD
can be difficult,11,12 but is important for adequate therapy, as TCRBCL is much more aggressive than LP HD.13
Useful markers may be epithelial membrane antigen (EMA) and CD80, for which the L&H cells in some cases of LP HD, but not the large neoplastic B cells in TCRBCL, are positive.6,14,15 In
addition, in LP HD, significant numbers of CD57-positive T cells, which can form rosettes around the L&H cells, and follicular dendritic cells
(FDCs) are found, whereas in TCRBCL, FDCs are absent and CD57+ T cells are rare.11,16
The differentiation stage of B cells can be studied by analysis of
rearranged V region genes. Naive IgM+IgD+ B
cells carry unmutated Ig gene rearrangements. In a germinal center (GC)
reaction, antigen-specific B cells clonally expand and diversify their
rearranged V genes by somatic hypermutation.17-19 Thus,
somatic mutations in rearranged V genes are found in GC B cells and
their descendents, ie, memory B cells and plasma cells. On this basis,
B-cell lymphoma precursors have been assigned to distinct stages of
normal B-cell differentiation (see Klein et al18 for review).
We recently analyzed V gene rearrangements in five subtypes of DLL,
including one case of TCRBCL, using DNA isolated from paraffin-embedded
tissues.20 Tumors of all five subtypes harbored mutated V
region genes and are thus derived from antigen experienced B cells.
However, as the template DNA was extracted from whole tissue sections
and polymerase chain reaction (PCR) products were directly sequenced,
we could not analyze the DLLs for intraclonal diversity, which may
allow a discrimination between a GC or memory B-cell derivation of the
tumor cells in TCRBCL. Here, we characterize the differentiation stage
of TCRBCL precursors by the analysis of Ig gene rearrangements in
single large CD20+ cells isolated by micromanipulation from
seven cases of TCRBCL.
| |
MATERIALS AND METHODS |
Tissues and clinical data.
Clinical data for the patients are given in Table 1. All biopsies were
performed for diagnostic purposes.
Immunostaining and EBV-encoded RNA (EBER) in situ
hybridization.
Immunohistochemistry with antibodies against CD20, CD15, CD30, CD3
(DAKO, Hamburg, Germany), and CD57 (Becton Dickinson, Heidelberg, Germany) was performed with the avidin-biotin-complex technique, alkaline phosphatase and Fast Red as substrate. The anti-FDC antibody DR53 was a gift from Dr G. Delsol (Toulouse, France). EBER
in situ hybridization was performed with paraffin-embedded tissue sections as described.21 The EBER probes were kindly
provided by Dr G. Niedobitek (Erlangen,
Germany)22 and digoxigenin-labeled using the DIG RNA
Labeling kit (Boehringer Mannheim, Mannheim, Germany).
Micromanipulation of single cells.
For micromanipulation, 5 to 10-µm sections of frozen biopsies were
stained with anti-CD20 or anti-CD3 antibody and alkaline phosphatase/Fast Red. Single cells were mobilized and aspirated with
micropipettes and the help of a hydraulic micromanipulator under a
microscope as described.23 In cases 1 to 4, L&H-like cells
not surrounded by smaller CD20+ cells were isolated. In
cases 5 to 7, the micromanipulated centroblast-like cells were often
surrounded by other CD20+ cells. Aspirated cells were
transferred to PCR tubes containing 20 µL of Expand High Fidelity PCR
buffer (Boehringer Mannheim) and stored at  20°C.
Single cell PCR.
Rearranged VH, V , and V genes
were amplified in a seminested approach, using family-specific primers
for the VH leader region and primers binding to sequences
within framework region (FR) I of human VH,
V, and V genes together with two sets of
nested primers for the joining (J) domains of each locus, as previously
described (the VHFRI 1 and 6 primers were not used in this
study).23-25 The primers used in the first round of PCR for
the amplification of V rearrangements were: V1 5'-GGTCCTGGGCCCAGTCTGTG-3', V2
5'-CAGTCTGCCCTGACTCAGCCT-3', V3a 5'-CTCAGCCACCCTCAGTGTCCGT-3', V3b
5'-CTCAGCCACCCTCGGTGTCAGT-3', V4
5'-TTTCTTCTGAGCTGACTCAGGAC-3', V6
5'-GAGTCTCCGGGGAAGACGGTA-3', V7
5'-GTGGTGACTCAGGAGCCCTCAC-3', V8
5'-ACTGTGGTGACCCAGGAGCCA-3', V9
5'-CCTGTGCTGACTCAGCCACCT-3', J1
5'-GCCACTTACCTAGGACGGTGAC-3', J23
5'-GAAGAGACTCACCTAGGACGGTC-3' and J67
5'-GGAGACT(C/T)ACCGAGGACGGTC-3'. The same V primers and
the following nested J primers were used in the second round of
amplification: J1i 5'-GGACGGTGACCTTGGTCCCAGT-3', J237i 5'-GACGGTCAGCTTGGT(G/C)CCTCC-3' and J6i
5'-GACGGTCACCTTGGTGCCACT-3'. Cells were digested with 0.25 mg/mL Proteinase K (Boehringer Mannheim) in 20 µL PCR buffer
for 1 hour at 50°C followed by a 10-minute incubation at 95°C.
The first round of amplification consisted of a 2-minute denaturation
step at 95°C, 4 minutes at 65°C, and 45 seconds at 72°C
followed by 34 cycles of 30 seconds at 95°C, 30 seconds at
61°C, and 45 seconds at 72°C. The first round was performed in
1x Expand buffer (Boehringer Mannheim) with 2 mmol/L MgCl2,
200 µmol/L of each deoxynucleoside triphosphate (dNTP), 0.04 µmol/L of each primer and 1 U of Expand enzyme mix (Boehringer Mannheim). One-microliter aliquots of the first round were used in the
second amplification round, where a separate PCR was performed for each
V gene family with the family-specific leader or FRI primer and a
nested J primer set. After a 2-minute denaturation at 95°C and 4 minutes at 65°C (VHFRI 2 and 5, V 1-6) or 67°C (VHL 1-5, V 1-4, and 6-9) or 69°C (VHFRI 3 and 4) and 45 seconds at
72°C, 39 cycles were performed at 95°C for 30 seconds, 61°C (VHFRI 2 and 5, V 1-6) or 63°C (VHL 1-5, V 1-4, and 6-9) or 65°C (VHFRI 3 and 4) for 30 seconds and 72°C for 45 seconds, in 1x PCR buffer (Boehringer Mannheim) with 1.5 (VHL 1 and 5, VHFRI 2-5, V 1-4, and 6-9) or 2.0 mmol/L MgCl2 (VHL 2-4, V 1-6),
200 µmol/L each dNTP, 0.125 µmol/L of each primer with the
exception of the JH primer set, which was used at a
concentration of 0.031 µmol/L and 0.7 U of Taq DNA polymerase
(Boehringer Mannheim).
To test whether the L&H-like cells contain several alleles of
rearranged Ig loci, 12 cells from case 4 were each digested in 60 µL
PCR buffer with 0.25 mg/mL Proteinase K and after extensive mixing
split into three 20-µL aliquots. After 10 minutes at 95°C, a
standard seminested PCR was performed for each aliquot.
Analysis of PCR products.
After agarose gel electrophoresis, PCR products were excised from the
gels, the DNA extracted with the QiaExII gel extraction kit (Qiagen,
Hilden, Germany) and directly sequenced using the dRhodamine or BigDye
Terminator cycle sequencing kits and an automated sequencer (ABI377,
Applied Biosystems, Weiterstadt, Germany). PCR products were sequenced
from both sides. Sequences were compared with the EMBL IMGT database
(http://www.genetik.uni-koeln.de/dnaplot/) and the GenBank data library
(release 98) using the GeneWorks software (Intelligenetics, Oxford, UK).
Fluorescence in situ hybridization (FISH).
Cytospin slides of isolated nuclei were prepared by mechanic
disaggregation of cyropreserved tumor tissue and subsequent pepsin digest as described recently.26 For chromosomes 7, 12, X
and Y, the indirectly-labeled centromeric probes D7Z1 (biotin-labeled) and D12Z3 (digoxigenin-labeled) (Oncor, Heidelberg, Germany) and the
directly-labeled probes CEPX and CEPY (Vysis, Stuttgart, Germany) were
used, respectively. Denaturation, hybridization, detection of
indirectly-labeled probes, and counterstaining was performed as
described.26 For each cytospin slide two probes were used for hybridization (D7Z1 together with D12Z3 and CEPX together with
CEPY). Hybridization signals were analyzed with a fluorescence microscope (Zeiss, Jena, Germany) and documented using the ISIS imaging
system (MetaSystems, Altrussheim, Germany).
| |
RESULTS |
Micromanipulation of single cells from TCRBCLs.
Single cells were micromanipulated from CD20-stained frozen tissue
sections of seven cases of TCRBCL (20 to 63 cells per patient, Tables 1 and
2). Criteria for selection of cells were
CD20 expression and size. In cases 1 to 4, the large micromanipulated
cells resembled L&H cells with few small CD20+ cells in the
background. In cases 5, 6, and 7, centroblast-like CD20+
cells were micromanipulated (Table 1). Here, the numbers of small
CD20+ cells in the background were higher than in cases 1 to 4, and L&H-like cells were not seen. In three cases, two
micromanipulation experiments were performed (Table 2). Aliquots of the
buffer covering the sections, and in case 2 also CD3+ cells
from an adjacent anti-CD3-stained section, were aspirated as negative
controls.
All large irregular shaped cells were CD3 ,
CD15, and CD30 in
immunhistochemistry and EBV-negative applying EBER-ISH (not shown). In
all cases, only a few scattered CD57+ T cells were found
and FDCs (DR53+ cells) were not seen.
PCR analysis of IgV gene rearrangements in the micromanipulated
cells.
V gene rearrangements for heavy (VH) and light chains
(VL) were amplified from single cells using sets of
family-specific V gene leader and/or FR I primers with two nested sets
of primers for the J regions, as described.23-25 In cases
in which amplifications with the VH leader
(VHL) and V primer sets did not yield products or only out-of-frame V rearrangements were
amplified, additional cells were analyzed using VH FRI
(VHFRI) and V primer sets. Amplification
efficiencies ranged from 39% to 78% (cells were counted as positive
when at least one V gene rearrangement was amplified). The negative
controls from the micromanipulations were included in a blinded
fashion; all were negative for V gene rearrangements (Table 2).
Clonality of the tumor cells in TCRBCL.
For cases 1 to 5, only clonal heavy and light chain rearrangements were
amplified (Table 2). In case 6, 34 of 40 amplified V gene
rearrangements for heavy and light chains belonged to four clonal
rearrangements (two VH and two V region
genes). Two VH and four V rearrangements
were unique. This indicates that in this case a small fraction of B
cells not belonging to the tumor clone was present in the lymph node.
Indeed, this case contained a considerable number of small B cells
besides the centroblast-like putative tumor cells (Table 1).
In case 7, clonal Ig gene rearrangements were neither detected by
single cell PCR nor by Southern blot analysis (data not shown).
Therefore, this case is not further considered here.
The large CD20+ cells in TCRBCLs harbor mutated,
clonal Ig heavy and light chain rearrangements.
All clonal VH genes and in-frame VL
rearrangements, as well as two VL out-of-frame
rearrangements amplified from single B cells of cases 1 to 6 were
mutated, with mutation frequencies ranging from 7.2% to 24% (average
frequency, 15.5%) for VH and 0.4% to 11.6% (average
frequency, 5.9%) for VL genes
(Table 3). From each of the cases, a
potentially functional VH and VL rearrangement was amplified. Three out-of-frame V and one out-of-frame V gene rearrangements were unmutated. The lack of
somatic mutation in the out-of-frame V gene
rearrangements of tumor cells that carry somatically mutated
VH and V genes is likely due to inactivation
of the loci by rearrangements using the Kappa deleting element (KDE),
which is frequently seen in human B cells.27-31 Because a
deleting element has not been described for the locus, it is
unclear why the out-of-frame V rearrangement in case 6 is unmutated despite the high load of mutations in the other rearrangements from the same tumor clone.
In three cases, V gene rearrangements that had most likely originally
been potentially functional were rendered nonfunctional by somatic
mutations. In the in-frame VH rearrangement of case 5, the
cysteine at position 92, which is thought to be important for proper
folding of the protein,32 is mutated to a tyrosine. In case
4, the mutated clonal in-frame V rearrangement is rendered nonfunctional by a stop codon. In addition, nearly the complete complementarity determining region (CDR) I is missing due to a
33-bp deletion, and the cysteine at position 88, which is important for
an intrachain disulfide bridge,32 is mutated to a serine.
However, case 4 also harbors a clonal in-frame V rearrangement, which is potentially functional and somatically mutated.
In case 6, two clonal in-frame VH rearrangements were
amplified. These two rearrangements were repeatedly amplified from the same single cells. While the rearrangement using VH4-34 is
potentially functional, the VH1-8 rearrangement contains
three stop codons and two insertions in FRIV, one of which resulted in
loss of the correct reading frame. The original rearrangement may have
been functional and inactivated by somatic hypermutation.
Intraclonal diversity in TCRBCLs.
In all six cases with clonal rearrangements, intraclonal diversity was
observed among the heavy chain rearrangements, and in four cases also
among the light chain sequences (Table 3, Fig 1). Usually, 2 to 3 sequence variants
of a clonal rearrangement were found. The greatest diversity was
observed for the potentially functional V rearrangement
in case 4 with six sequence variants. Most of the sequence variants are
due to single nucleotide exchanges and were amplified several times.
None of these diversifying point mutations in the potentially
functional rearrangements rendered the genes nonfunctional. In cases 2 and 4, besides nucleotide exchanges, also deletions, and in case 3, a
duplication were observed in several cells. Whereas in cases 2 and 4, the deletions abolished the coding capacity of these rearrangements,
the 21-bp duplication in the VH CDRIII of case 3 preserves
the correct reading frame.
View larger version (25K):
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| Fig 1.
Intraclonal diversity in TCRBCL. Genealogical trees for
the potentially functional rearrangements with intraclonal diversity.
Letters and figures in the rectangles are the designations of the
germline genes with the greatest homologies. Assumed intermediates in
the genealogical trees are designated with X and Y. Figures in the
circles indicate how often a specific sequence variant was found.
Beside the lines connecting the sequence variants, positions of
nucleotide exchanges and the new nucleotides are given. Mixed clonal
sequences are excluded from this figure.
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Analysis of mutation pattern.
The ratio of replacement (R) to silent (S) mutations in the FRs of
functional V gene rearrangements can give hints whether cells have been
under selective pressure for expression of an antigen receptor. In that
case, R mutations are counterselected in the FRs to preserve the
evolutionary optimized structure of the antibody V domain. In the
absence of selection, like in out-of-frame rearrangements, R mutations
in the FRs are not counterselected.
Indeed, the R/S ratios of the mutations within the FRs in productive
rearrangements of various B-cell subsets with mutated V gene
rearrangements have been shown to be in the range between 1.0 and 1.6, whereas for a collection of nonfunctional out-of-frame rearrangements,
the ratio is 3.0, the value expected assuming random
mutagenesis.18,20 For the 190 mutations in the FRs of the
12 potentially functional rearrangements in the six cases of TCRBCL
analyzed, a R/S ratio of 1.5 was found, which is within the range
typical for antigen-selected cells.
A R/S analysis was also performed for two other in-frame rearrangements
(VA2 of case 4 and VH1-8 of case 6), which
might have been functional before they accumulated inactivating
mutations. Indeed, these rearrangements show R/S ratios of 1.5 and 2.0, respectively, indicating that they had been positively selected before inactivation.
The large CD20+ cells in TCRBCL are polyploid and
contain different sequence variants of the same clonal V gene
rearrangement within a single cell.
From several single tumor B cells, mixed sequences representing two
variants of the same clonal rearrangement were obtained, indicating the
presence of two or more copies of the clonal V gene rearrangement in
the respective cells. These mixed sequences were mainly due to single
nucleotide differences, and in one case, to two deletions
(V1g rearrangement of case 4). Such mixed sequences were
found at least once in each case. In half of the cases in which a mixed
sequence was detected, it was seen in several single cells (Table 3).
To verify the existence of multiple copies of rearranged V genes in
large CD20+ cells, the DNA of single micromanipulated cells
from one case (case 4) was split into three aliquots and the standard
seminested amplification protocol was performed for each of the
aliquots (see Materials and Methods). Five clonal VH and
five clonal V rearrangements were amplified from 12 cells. From two aliquots of one cell, an identical clonal
V gene was amplified. From two aliquots of another cell,
clonal VH rearrangements differing at two nucleotide
positions were obtained. This confirms that at least in case 4 multiple
copies of the same clonal rearrangement, which may show sequence
variation, are present within some or all tumor cells.
The existence of multiple copies of clonal rearrangements could be due
to duplications of the rearrangements, gain of single chromosomes 2, 14, or 22 carrying the V gene loci or a gain of a whole chromosome set
leading to polyploidy. The latter two possibilities can be
distinguished by FISH using probes for chromosomes other than those
carrying the V genes. Thus, FISH was performed on nuclei isolated from
frozen tissues of cases 1 to 3 using probes for chromosomes 7, 12, X
and Y. In all three cases, additional signals for chromosomes 7, 12, and the gonosomes were seen in a significant percentage (5% to 10%)
of interphase nuclei evaluated. Thus, the results of the FISH study
suggest polyploidy and render the presence of isolated gains of the
chromosomes carrying the V genes unlikely. As the proportion of the
large CD20+ cells in the three tissues analyzed is about
10% to 15%, it is likley that the large CD20+ cells
represent the polyploid cells. Thus, the large CD20+ cells
in TCRBCL contain abnormal numbers of some (and potentially all) chromosomes.
| |
DISCUSSION |
Clonality of the L&H- or centroblast-like cells in TCRBCL.
From six cases of TCRBCL, clonal Ig gene rearrangements were amplified
from single micromanipulated CD20+ cells of irregular size.
This confirms that in TCRBCL the irregular L&H-like cells (cases 1 to
4) or the centroblast-like cells (cases 5 and 6), assumed to be the
neoplastic cells, indeed represent clonal B-cell populations. The
amplification of five unique rearrangements in case 6 shows that also
nonclonal infiltrating B cells are present at least in this TCRBCL and
sometimes may be difficult to distinguish from the neoplastic cells.
TCRBCL are derived from GC B cells.
All clonal VH and in-frame VL gene
rearrangements were mutated with mutation frequencies ranging from
0.4% to 24%. These data are in agreement with those obtained for the
one case analyzed previously20 and show that the tumor
cells are derived from mature B cells that had participated in a GC
reaction (ie, GC or post-GC B cells). Moreover, because the process of
somatic hypermutation appears to be restricted to GC B cells, the
finding of intraclonal diversity in all six cases identifies GC B cells as the precursors of the tumor clone in TCRBCL. However, it is unclear
whether the somatic hypermutation machinery was still active in the
tumor cells when the biopsies were taken. It is likewise possible that
the observed intraclonal diversity resulted from ongoing mutation at a
very early stage of tumor clone expansion and that subclones generated
in this way were then stably propagated. A GC B cell derivation of
TCRBCL is further supported by the expression of the GC B cell-specific
transcription factor bcl-6 by 10% to 90% of the lymphoma
cells33,34 (indeed, bcl-6 was expressed by the tumor cells
in all six cases analyzed in the present study; data not shown) and the
centroblast-like morphology of the large irregular cells in some cases.
The somatic mutations in the rearranged V genes were not restricted to
nucleotide exchanges, but also included duplications, insertions, and
deletions (Table 3 and Fig 1). The occurrence of duplications,
insertions, and deletions in somatically mutated V region genes is in
accord with the work of Goossens et al35 and Wilson et
al36 who recently showed that such events occur during the
process of somatic hypermutation.
For each of the six cases, potentially functional Ig heavy, as well as
light chain, genes were amplified. The average R/S ratio of 1.5 for the
FRs of those rearrangements is in the range that is typical for normal
antigen-selected B cells.18 This indicates that, in
general, the precursors of the tumor clones were selected for a
functional antigen receptor.
Crippling mutations in TCRBCL?
Despite those clear signs for antigenic selection in the tumor
precursors, in several of the cases, potentially crippling mutations
were observed in clonal V gene rearrangements. In case 2, seven of 15 in-frame VH gene sequences were rendered nonfunctional by a
19-bp deletion. In case 4, a fraction of the sequences harbored two
deletions, one of which resulted in loss of the correct reading frame.
In addition, it is conceivable that nearly half of the sequences of the
VH4-31 rearrangement in case 3 are no more able to bind to
the initial antigen recognized by the B-cell receptor, because of a
21-bp duplication in CDRIII. In case 5, the cysteine at codon 92 is
mutated to tyrosine in all clonal VH rearrangements amplified. This cysteine is thought to be important for correct folding
of the protein.32 Thus, it is possible that in some cases
of TCRBCL, the tumor cells or subpopulations of them lost dependence on
expression of a functional antigen receptor. However, the following
aspects are important in evaluating the "crippling" mutations:
(1) it has been described that a hybridoma expressed a functional
antigen receptor despite a cysteine 92 to tyrosine mutation.37 Therefore, mutations of the conserved cysteine
92 are not necessarily crippling. (2) In case 4, a mixed sequence of
the V gene rearrangement with and without the two
deletions was amplified from six cells (Table 3). Thus, these tumor
cells most likely contained two copies of the clonal V
gene rearrangement, one of which was still functional. This may well be
true for all cells of this tumor, given the limited efficiencies of V
gene amplification from single, micromanipulated cells. That the tumor cells in TCRBCL often contain more than two copies of several (or even
all) chromosomes is supported by the FISH analysis, which showed
additional copies of the chromosomes investigated, ie, chromosomes 7, 12, and the gonosomes, in the three cases analyzed. Moreover, it has
been reported that 91% of DLLs show numerical chromosomal
aberrations.38
Thus, in TCRBCL, single tumor cells may contain two or more copies of a
V gene rearrangement and between these copies there may be sequence
variation. In case 2, the functional and crippled variants of the
VH gene rearrangement were never amplified together from a
single cell. Thus, in this case, a subclone of the tumor cells might
have lost the capacity to express a functional antigen receptor.
However, because the PCR efficiency and the total number of sequences
was lower in this case than in case 4, it is possible that the tumor
cells harbor two copies of the VH gene rearrangement (one
functional and one crippled) also in this case.
Indications for V gene editing in human GCs.
In two instances we observed crippling mutations in tumor cell-derived
V gene rearrangements, which are indicative of secondary V gene
rearrangements (receptor editing) in the course of the GC
reaction.39,40 In case 4, an in-frame V gene
rearrangement was rendered nonfunctional by a stop codon within the
V gene segment and a 33 bp deletion in CDRI.
Interestingly, the tumor cells also harbor a potentially functional
V gene rearrangement. Conceivably, the tumor precursor,
a GC B cell, initially expressed a light chain gene and when this
rearrangement was crippled by the stop codon and/or the deletion, it
successfully performed a productive V gene
rearrangement. The considerably lower mutation load in the
V gene (1.4% to 4%) compared with the VH,
as well as V gene (8.9% and 6.9%, respectively) is in line with the interpretation that the V gene
rearrangement occurred in the course of the GC reaction, so that it
went through less rounds of somatic mutation than the two other V
genes. In addition, the R/S ratio of 1.5 for mutations in the FRs of
the V rearrangement indicates that the gene was
initially selected and hence expressed.
In case 6, two clonal in-frame VH gene rearrangements were
amplified. One of the rearrangements had been inactivated by somatic mutation. These two in-frame VH rearrangements could have
been generated during B-cell development, and somatic mutation could then have inactivated one rearrangement of a potential double producer.
The expression of two functional heavy chain genes has indeed been
described for a fraction of cases of B-cell chronic lymphocytic
leukemia (B-CLL).41 However, it is also
possible that the tumor precursor contained initially only one
functional VH rearrangement, which was inactivated in the
GC by somatic mutation and the second VH in-frame
rearrangement occurred thereafter. The R/S ratio of 2 for the mutations
within the FRs of the inactivated rearrangement may reflect such a
partial selection for functionality.
The relationship between TCRBCL, other B-cell non-Hodgkin's lymphoma
(NHL) and HD.
Other types of DLLs also harbor mutated Ig gene rearrangements and,
based on the analysis of a collection of cases from different types of
DLLs (10 centroblastic lymphomas, 5 mediastinal sclerosing lymphomas, 2 immunoblastic lymphomas, one large cell anaplastic lymphoma, and one
TCRBCL), the tumor precursors appear to be selected for expression of a
functional antigen receptor also in these other entities.20
However, it is unclear whether the other DLLs are also derived from
mutating GC B cells. Whereas ongoing mutation was not observed in 17 cases analyzed, in one study,42 ongoing mutation has been
described in a centroblastic lymphoma20 and four DLLs not
further specified.43,44
In terms of the differentiation stage of the tumor precursors, TCRBCL
appears to be closely related to follicular lymphoma45 and
LP HD25,46,47 in which the consistent finding of ongoing mutation indicates a derivation from mutating GC B cells as well. Moreover, intraclonal diversity of rearranged V genes is occasionally also seen in sporadic and endemic Burkitt's lymphoma48,49
and mucosa-associated lymphoid tissue (MALT)
lymphomas.50 Thus, although the precursors of all these
lymphomas derive from the same B-cell compartment, they give rise to
different types of lymphomas. It is likely that GC B-cell-derived
lymphomas differ in their dependence on antigenic triggering, in the
transforming events, and the subsets of GC B cells that are subject to
the initial transforming events.18
The diagnostic distinction of TCRBCL from the diffuse variants of LP HD
is often difficult, but important for adequate therapy, as both
entities show a strikingly different clinical behavior, TCRBCL being
much more aggressive than the clinical indolent LP HD.13 In
LP HD, the tumor cells seem to be dependent on a GC-like microenvironment with FDCs and CD57+ T cells, while in
TCRBCL, no GC remnants are found.11,16 Comparison of the V
gene rearrangements and their mutation pattern in TCRBCL and LP
HD25 does not show a clear distinction between the two diseases: (1) both types of tumor cells harbor highly mutated clonal Ig
gene rearrangements (with average VH mutation frequencies of 10.1% for LP HD and TCRBCL) and in all cases analyzed, potentially functional in-frame rearrangements were amplified for Ig heavy and
light chains.18,25 (2) The mutation pattern indicates that in both entities the tumor precursors are generally selected for the
expression of a functional antigen receptor, in distinction to
classical HD, where the tumor as a rule appears to be derived from
crippled GC B cells.24 (3) Although the interpretation of
the crippled rearrangements in two cases of TCRBCL of the present study
is ambigious, it appears that in LP HD, as well as in TCRBCL, subclones
of the tumor may occasionally lose dependence on antigen receptor
expression, resulting in the outgrowth of subclones harboring crippling
mutations.46,51
The only molecular distinction between TCRBCL and LP HD that became
evident from the present study is the consistent finding of two or more
copies of clonal rearrangements showing mixed sequences within single
cells in each of the six cases of TCRBCL, while this was only rarely
observed in LP HD (two of 27 sequences in one of five cases,
unpublished observation). The consistent finding of such mixed
sequences in TCRBCL indicates that chromosomal aneuploidy is a frequent
feature of the tumor cells in this disease and that the hypermutation
machinery is still active when or after chromosomes have been
duplicated. The low frequency of mixed sequences in the cases of LP HD
so far analyzed25 suggests that numerical chromosomal
abnormalities involving the chromosomes harboring the Ig loci are
either rare in LP HD or that the hypermutation machinery was no longer
active when such hypothetical chromosomal duplications happened.
Taken together, the present study identifies mutating GC B cells as the
precursors of the tumor clones in TCRBCL, adding this disease to the
large collection of GC B-cell-derived malignancies and showing a close
relationship between this lymphoma and LP HD at the level of the tumor precursors.
| |
ACKNOWLEDGMENT |
We thank Christiane Gerhard and Tanja Schaffer for excellent technical
assistance. We are grateful to Holger Kanzler for critically reading
the manuscript.
| |
FOOTNOTES |
Submitted August 11, 1998; accepted November 23, 1998.
Supported by the Deutsche Forschungsgemeinschaft through SFB 502, the
Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung, and the
Wilhelm-Sanders-Stiftung. B.S. holds a Hermann and Lilly Schilling professorship.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Andreas Bräuninger, PhD,
Department of Pathology, University of Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt, Germany; e-mail: Braeuninger{at}em.uni-frankfurt.de.
| |
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TOP
ABSTRACT
INTRODUCTION