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Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3857-3864
VH Gene Analysis of Primary Cutaneous B-Cell Lymphomas: Evidence for
Ongoing Somatic Hypermutation and Isotype Switching
By
W.M. Aarts,
R. Willemze,
R.J. Bende,
C.J.L.M. Meijer,
S.T. Pals, and
C.J.M. van Noesel
From the Department of Pathology, Academic Medical Center, Amsterdam,
The Netherlands; and the Departments of Dermatology and Pathology, Free
University Hospital, Amsterdam, The Netherlands.
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ABSTRACT |
Primary cutaneous B-cell lymphomas are B-cell non-Hodgkin's
lymphomas that arise in the skin. The major subtypes discerned are
follicle center cell lymphomas, immunocytomas (marginal zone B-cell
lymphomas), and large B-cell lymphomas of the leg. In this study, we
analyzed the variable heavy chain (VH) genes of 7 of these lymphomas,
ie, 4 follicle center cell lymphomas (diffuse large-cell lymphomas) and
3 immunocytomas. We show that all these lymphomas carry heavily mutated
VH genes, with no obvious bias in VH gene usage. The low ratios of
replacement versus silent mutations observed in the framework regions
of 5 of the 7 lymphomas suggest that the structure of the B-cell
antigen receptor was preserved, as in normal B cells that are selected
for antibody expression. Moreover, evidence for ongoing mutation was
obtained in 3 immunocytomas and in one lymphoma of large-cell type. In addition, in 1 immunocytoma, both IgG- and IgA-expressing clones were
found, indicative of isotype switching. Our data provide insight into
the biology of primary cutaneous B-cell lymphomas and may be of
significance for their classification.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
PRIMARY CUTANEOUS B-cell lymphomas
(PCBCLs) are defined as B-cell non-Hodgkin's lymphomas (B-NHLs) that
arise in the skin, with no evidence of extracutaneous disease for a
period of at least 6 months after diagnosis. PCBCLs generally have a much better prognosis than nodal B-NHLs of comparable histologic subtype.1-3 The European Organization for Research and
Treatment of Cancer (EORTC) has proposed a new classification for
PCBCLs, based on clinical, histological, and immunological
criteria.3 The major subtypes are primary cutaneous
follicle center cell lymphomas and immunocytomas (marginal zone B-cell
lymphomas), both of indolent clinical behavior. Another group of
PCBCLs, ie, large B-cell lymphomas of the leg, is considered to be of
intermediate malignancy. Two provisionally designated entities are
intravascular large B-cell lymphomas and plasmacytomas.
Although the nomenclature of PCBCLs suggests an established
relationship with nodal B-NHL counterparts and/or with
differentiation stages of normal B-cell ontogeny, virtually no data are
as yet available on the composition of the B-cell antigen receptors
(BCR) of PCBCLs. Normal B-cell maturation is characterized by stepwise alterations of the BCR. Naive B cells carry unmutated Ig variable heavy
chain and light chain genes that are expressed at the cell surface as
IgM and IgD isotypes.4,5 In germinal centers of secondary
follicles, B cells proliferate and compete to bind antigens that are
exposed at the surface of follicular dendritic cells (FDCs).6 Recognition of antigen elicits signals essential
for proliferation and differentiation. During the subsequent cell divisions, somatic mutations are introduced in the variable Ig genes
(reviewed by Kocks and Rajewsky7). Because of strict selection processes, the germinal center reaction finally yields B
cells with nonrandom patterns of somatic mutations and augmented affinity for the recognized antigens.8 These post-germinal center cells, either memory B cells or plasma cells, often express heavy chain isotypes other than µ and  ,4,8 which has
implications for the effector functions of the secreted Ig.
To obtain information on the maturational state of PCBCLs, we analyzed
the variable heavy chain (VH) region genes of 7 of these lymphomas. We
show that they all carry significantly mutated VH genes with mutation
patterns reminiscent of antigen selection processes. This finding
indicates that PCBCLs are derived from germinal center cells or their
descendents. Moreover, evidence was obtained for ongoing somatic
hypermutation and isotype switching, features that are shared with
extracutaneous B-NHLs of mucosa-associated lymphoid tissue (MALT) and
follicular lymphomas.
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MATERIALS AND METHODS |
Patient material.
Tissue material of 7 PCBCLs, ie, 4 follicle center cell lymphomas, 3 immunocytomas (marginal zone B-cell lymphomas), and 1 pseudolymphoma,
was obtained from the Departments of Dermatology and Pathology of the
Free University Hospital and the Department of Pathology of the
Academic Medical Center (Amsterdam, The Netherlands). The diagnoses
were based on the characteristic clinical and histologic criteria,
described previously.3 The follicle center cell lymphomas showed a predominance of centroblasts and are further referred to as
diffuse large B-cell lymphomas. Follow-up data confirmed the favorable
prognosis of these lymphomas, with all patients alive and in complete
remission 12 to 106 months after diagnosis. The clinical and
histological data of the 8 patients are summarized in
Table 1.
Immunohistochemistry.
The expression of surface Ig isotype, CD70, and the presence of FDCs
was determined immunohistochemically on cryostat sections. Monoclonal
antibodies specific for human Ig isotypes and CD21L (DRC-1) were
purchased from DAKO (Glostrup, Denmark), except for anti-IgM, which was
obtained from Becton Dickinson (Erembodegem-Aalst, Belgium).
CLB-CD70/1, specific for CD70, was a kind gift from Dr R.A.W. van Lier
(Central Laboratory of The Netherlands Red Cross Blood Transfusion
Service, Amsterdam, The Netherlands). Acetone-fixed tissue sections
were preincubated with 10% normal goat serum (Sera Lab, Sussex, UK) in
phosphate-buffered saline (PBS) for 15 minutes. After 1 hour of
incubation with the primary antibody, endogenous peroxidase was blocked
for 10 minutes with 0.1% NaN3, 0.3%
H2O2 in PBS. Subsequently, sections were
incubated with biotin-conjugated rabbit antimouse (Dako) for 30 minutes. After incubation with a streptavidin-biotin-peroxidase complex (Dako) for 30 minutes, horseradish peroxidase activity was detected with 3-amino-9-ethylcarbazole (Sigma, St Louis, MO) and 0.03% H2O2. Sections were counterstained with
haematoxylin (Merck, Darmstadt, Germany).
RNA isolation and cDNA synthesis.
Total cellular RNA was isolated from frozen tissue sections using the
TRIZOL reagent (Life technologies, Breda, The Netherlands) according to
the manufacturer's instructions. For cDNA synthesis, 10 µg
of RNA was incubated with 5 nmol of pd(N)6 primer
(Pharmacia Biotech, Roosendaal, The Netherlands) for 10 minutes at
65°C. After cooling on ice, the reaction mixture was added to a
final volume of 50 µL. It contained 400 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Life Technologies,
Breda, The Netherlands), 8 mmol/L dithiothreitol (DTT), 1 mmol/L of each dNTP, 1× first-strand buffer (50 mmol/L
Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl2), and 60 U
of RNAse inhibitor (Boehringer Mannheim, Almere, The Netherlands). The
reaction was performed for 1 hour at 37°C. Subsequently, the enzyme
was inactivated during 10 minutes at 95°C.
Polymerase chain reactions (PCRs).
The complementarity determining region 3 (CDR3) was amplified using a
forward primer with specificity for framework region 3 (FR3) in
combination with reverse primers specific for JH (JHseq), Cµ, C
(C2), C , or C (Table 2). Either, 1 µL of the cDNA reaction mixture was used or (for a nested PCR) 1 µL
of PCR product from a VH family-specific PCR was used. The PCR mixture
contained 1× Taq buffer (20 mmol/L Tris-HCl, 50 mmol/L KCl, pH
8.4), 0.2 mmol/L of each dNTP, 1.5 mmol/L MgCl2, 2 U of Taq polymerase (Life Technologies), and 0.5 µmol/L of each
primer. First, 10 cycles of amplification were performed in the thermal
cycler (PTC-100; MJ Research Inc, Watertown, MA), ie, successively 30 seconds at 95°C, 20 seconds at 57°C, and 20 seconds at
72°C. The next 40 cycles of amplification consisted of 30 seconds
at 95°C, 20 seconds at 55°C, and 20 seconds at 72°C. The
reaction was completed for 6 minutes at 72°C. PCR products were
analyzed on a 3% Metaphor agarose gel (FMC Bioproducts, Rockland, ME).
For the VH family-specific PCR, reactions were performed with one of
the VH family-specific leader primers (Table 2), combined with the
appropriate reverse primer, either JH, Cµ, C, or C. The PCR
reaction mixture was the same as for the CDR3-specific PCR, except that
1 U of Taq polymerase and 0.25 µmol/L of each primer was used. Thirty
cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C were performed. The reaction was terminated for 6 minutes at 72°C. The PCR products were analyzed on a 1% standard
agarose gel (Sigma).
Cloning and sequencing of PCR products.
After excision of the PCR products from an agarose gel and isolation of
DNA with the Qiaex kit (Qiagen, Hilden, Germany), the PCR products were
ligated into pGEM-T vectors (Promega, Leiden, The Netherlands),
according to the manufacturer's instructions, and transformed into
DH10b bacteria (Life Technologies). Subsequently, both strands of the
inserts were sequenced from 4 or more colonies to obtain the sequence
of the dominant clone, the consensus sequence. Sequencing was performed
with an ABI sequencer (Perkin Elmer Corp, Norwalk, CT) using the
dye-terminator cycle-sequencing kit (Perkin Elmer Corp), according to
the manufacturer's instructions. To determine the Taq error rate of
our experimental design, 19 clones of CD79a and CD79b were sequenced.
These clones were generated according to the same PCR and cloning
procedures as used for the VH genes. The Taq error frequency thus
established is 0.14%, which amounts to 0.4 mutation/VH clone.
Assignment of mutations.
The seqences found were compared with published germline sequences,
using the Vbase database9 and DNAplot on the Internet (http://www.genetik.uni-koeln.de/dnaplot; programmed by H.H. Althaus) to identify mutations. Mutations at the last nucleotide position of the
V gene were excluded from the mutational analysis, because they might
result from nucleotide deletions at the joining sites. To calculate
whether the excess or scarcity of replacement mutations in the FRs had
occurred by chance, we used the binomial distribution model as proposed
by Chang and Casali.10 In B cells selected for antibody
expression, there is a counterselection for replacement (R) mutations
in the FR to maintain the structure of the antibody. The ratios of
replacement versus silent (R/S) mutations in the CDRs are often higher
than expected. However, the R/S values found in the CDRs cannot be used
as arguments for or against antigenic selection. Dörner et
al11 showed that the R/S values of both FR and CDR were
higher in nonproductive and therefore unselected rearrangements than in
productive, antigen-selected rearrangements. Also, it can be imagined
that, in an already selected Ig with optimal affinity, additional
replacement mutations in the CDRs are unfavorable.
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RESULTS |
Morphology of PCBCLs.
The clinical and histological data of the 7 PCBCLs analyzed are
summarized in Table 1. The group comprised 4 follicle center cell
lymphomas (all diffuse large B-cell lymphomas) and 3 immunocytomas (marginal zone B-cell lymphomas). In addition, a lesion showing the
clinical and histologic features of a pseudolymphoma was included in
this study (no. 8). Despite the fact that all lymphomas displayed a
diffuse growth pattern, areas of FDCs were detected in 5 of the 7 lymphomas (nos. 1, 2, 3, 5, and 6). In PCBCLs no. 5 and 6, extensive
ill-defined networks of FDCs were found
(Fig 1), which suggests that the FDCs form
an integral part of this neoplasm. Interestingly, CD70 expression was
also found only in lymphomas no. 5 and 6 (not shown). In the other
cases (no. 1, 2, and 3), the FDC clusters were more or less nodular and
well circumscribed. Here, it is unclear whether they belong to the
tumors or represent the remains of pre-existent reactive follicles that
were infiltrated by the neoplastic B cells. These different
patterns of FDCs in PCBCL have also been reported by Mori et
al.12
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| Fig 1.
Tissue section of lymphoma no. 5 stained for FDCs with
antibodies against CD21L (DRC-1). Magnification (A) × 50 and (B) × 125.
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Ig isotype expression.
The Ig heavy chain isotype of the 7 PCBCLs studied was determined
immunohistochemically (Table 1) and by PCR, ie, by amplifying the CDR3
region with an FR3 primer in combination with primers specific for the
5 regions of Cµ, C (C2), C, or C heavy chains (Table 2). Because of differences in length of the diversity (D)
genes13 and random nucleotide additions by terminal
deoxytransferase14 at junctions of the V-D and D-J gene
segments, the size of the CDR3 regions varies considerably between
B-cell clones.15 Therefore, a CDR3-specific PCR on a
polyclonal B-cell population yields products of variable size, visible
as multiple bands or a smear pattern after electrophoresis, whereas
this PCR on a clonal population yields a single band
(Fig 2). In our hands, this CDR3-specific PCR has proven very useful as a screening assay and as a sensitive method to identify the lymphoma-derived Ig when, due to the copresence of nonneoplastic B cells, multiple VH genes are amplified from a tissue
specimen. With respect to the assessment of the Ig isotype expression,
either immunohistochemically or by PCR, we encountered no discrepancies
(compare Tables 1 and 3). For example, in
lymphoma no. 6, clear membrane expression of both IgM and IgD was
detected by immunohistochemistry (Table 1). As expected, products of
single length were amplified by PCR using the FR3 primer in combination with JHseq, Cµ, and C primers, whereas smears were obtained with C2 and C as downstream primers (Fig 2, left panel). PCR of a pseudolymphoma yielded products of variable lengths in all lanes (Fig
2, right panel). In 4 PCBCLs (no. 2, 4, 5, and 7), the two methods to
assign the Ig isotype were complementary (Tables 1 and 3);
immunostaining of lymphoma no. 5 showed clear membrane expression of
IgM and, at most, very weak expression of IgD. However, using PCR, both
IgM and IgD were clearly amplified from this lymphoma. Also, in cases
no. 2 and 7, the Ig isotypes used could only be established by PCR.
Alternatively, lymphoma no. 4 was found to express both IgM and IgD
immunohistochemically, whereas by PCR only IgM expression was found.
This was possibly due to the low amount of cDNA available from this
lymphoma. In summary, 4 lymphomas (no. 3, 4, 5, and 6) were found to
coexpress IgM and IgD, whereas 3 lymphomas (no. 1, 2, and 7) expressed
IgG. Interestingly, the CDR3-specific PCR of lymphoma no. 7 yielded
single bands in the lanes corresponding to the JHseq, C2, and C
primers, which is suggestive of the presence of clonal populations of
both IgG and IgA isotypes (Fig 2, middle panel). To analyze whether the
IgG and IgA products originated from the same tumor clone, the VH genes
were amplified with VH family-specific primers in combination with the
constant C or C primers. Subsequent sequencing of the VH-C and
VH-C PCR products confirmed that they harbored the same VDJ
rearrangement (not shown).
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| Fig 2.
PCR analysis on the CDR3 region of lymphomas no. 6 (left
panel) and 7 (middle panel) and the pseudolymphoma (right panel). The
FR3 primer was used as upstream primer, and JHseq, Cµ, C2, C,
and C primers were used as downstream primers, as indicated above
the lanes.
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VH, D, and JH gene usage.
VH genes were amplified with family-specific VH leader primers (Table
2). On the PCR products thus obtained, we performed a nested
CDR3-specific PCR to confirm that a particular VH product originated
from the clonal population (data not shown). Subsequently, the VH
product was cloned and sequenced. The nucleotide sequences have been
deposited at the GenBank database (accession nos. AF052379 through
AF052386). The VH sequences were compared with the germline genes with
the highest homology and, accordingly, the number of somatic mutations
was determined (Table 3). Four PCBCLs used genes of the VH3 family,
whereas VH1, VH4, and VH5 family genes were each found once. Comparison
with germline JH gene segments showed that the JH4b gene was present in
4 rearrangements (lymphomas no. 1, 3, 5, and 7). The lymphomas no. 2, 4, and 6 used the JH6c, JH3b, and the JH5b genes, respectively. Corbett
et al13 proposed stringent criteria for the assignment of D
genes: at least 10 consecutive nucleotides of identity are required to
confidently assign a D gene segment. According to these criteria, we
could only assign the D21-9 and DXP4 gene segments of lymphomas no. 4 and 6, respectively.
Mutation patterns.
All lymphomas expressed extensively mutated VH genes, ranging from 15 to 52, with an average of 28.6 mutations per VH sequence (Table 3).
Within our limited set of 7 PCBCLs, there was no obvious difference in
the number of mutations between IgM+IgD+ or
IgG+ lymphomas. Analysis of the distribution of replacement
(R) versus silent (S) mutations10 demonstrated that 5 of
the 7 PCBCLs (no. 2, 3, 4, 5, and 6) contained a significantly lower
number of R mutations in the FRs (Table 4)
than would be expected if mutations had occurred by chance alone, ie,
in the absence of selective forces. Except for lymphoma no. 6, the R/S
values within the CDRs were always higher than those within the FRs of
the corresponding VH genes.
Intraclonal variation.
Information on the intraclonal variation was obtained by sequencing of
each lymphoma at least four amplified VH molecules (Table 3 and
Fig 3). No differences were found in the
sequences of the individual clones of lymphomas no. 1 or 2, which are
both IgG-expressing PCBCLs. Also, in lymphoma no. 3, which coexpresses IgM and IgD, no intraclonal variation was observed. However, lymphomas no. 4, 5, and 6 and the IgG-clone of no. 7 showed intraclonal variations of 0.6, 1.4, 2.6, and 3.4 mutations/clone, respectively. Moreover, in each of these lymphomas, mutations were found that were
shared by more than one clone (Fig 3), which is strong evidence for
ongoing somatic hypermutation rather than Taq error. Lymphomas no. 4, 5, and 6 coexpressed IgM and IgD (Tables 1 and 3), as determined by
immunohistochemistry and PCR. The highest level of intraclonal
variation was observed in the IgG clone of lymphoma no. 7, demonstrating that ongoing mutation is not necessarily confined to
IgM+ PCBCLs. However, in contrast to the high mutation
frequency of the IgG+ tumor cells, the intraclonal
variation found in the IgA+ tumor cells amounted 0.2 mutations/clone, which does not exceed the Taq error frequency of
0.14% (~0.4 mutation/clone). Yet, the consensus sequences of the
IgG- and the IgA-expressing subclones were identical.
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| Fig 3.
Intraclonal variation found in lymphomas no. 4, 5, and 6 and the IgG-clone of no. 7. Indicated are only the codons in which
mutations were found. The first clone of each lymphoma is the consensus
sequence. The mutations compared with this consensus sequence are
shown. Replacement mutations are shown in capitals, and silent
mutations are shown in small letters.
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DISCUSSION |
In this study, the VH genes of 7 PCBCLs were analyzed to learn what
maturation steps were traversed by the tumor cells and to gain insight
into the biological relation with other B-NHL. The PCR method used here
has proven to be a fast and reliable approach to establish clonality at
molecular level, despite the facts that only small amounts of frozen
tissue were available and that the lymphomas harbored heavily mutated
VH genes. To assay clonality with this method, 1 to 2 µg of RNA is
sufficient, which is obtainable from 1 to 2 mg of lymphoma tissue. For
comparison, for Southern blotting at least 30 µg genomic DNA is
required, which equals 6 or 7 mg of tissue.
Within this limited set of PCBCLs, no bias in the use of VH genes was
observed. Four of the lymphomas expressed VH3 genes (no. 2, 3, 4, and
5), whereas VH1, VH4, and VH5 genes were each found once (no. 7, 6, and
1, respectively). In normal peripheral B cells, VH3 genes are used in
56%, the VH4 and VH1 genes in 20% and 13%, respectively, whereas
VH2, VH5, and VH6 are each found in less than 10%.16-18 In
our study, 3 of the 4 large-cell PCBCLs use different members of the
VH3 family. For nodal diffuse large B-cell lymphomas, one group
reported that the VH4 gene was expressed in 15 of 17 cases
studied.19 However, a more recent study observed no bias in
VH gene usage within a set of 18 extracutaneous diffuse large B-cell
lymphomas.20
The mutation frequency in the VH genes of the 7 PCBCLs studied here
ranged from 15 to 52, with an average of 28.6 mutations (10%). This is
significantly higher than the number of mutations found in normal B
cells: for normal germinal center and memory B cells, Pascual et
al4 reported average numbers of mutations of 5.7 (2%) and
9.5 (3%) in IgM- and IgG-derived VH sequences, respectively. Similar
numbers of mutations were found by Tomlinson et al,21 ie,
an average of 3.7 (1%) mutations per IgM-derived sequence and 10.2 (3%) mutations per IgG-derived sequence. We found high numbers of
somatic mutations in both IgM-positive and IgG/IgA-positive PCBCLs,
either of small- or large-cell types, without gross quantitative
differences. Interestingly, all 4 IgM+ PCBCLs also
coexpressed IgD (no. 3, 4, 5, and 6; Tables 1 and 3) and carried
significantly mutated VH genes with low R/S ratios in the FRs. This
contrasts with normal naive IgM+IgD+ B
cells4 as well as with mantle cell lymphomas22
and a subset of IgM+IgD+ B-cell chronic
leukemias23 that are unmutated. It is possible that, in
lymphomas no. 3, 4, 5, and 6, transformation from normal B cells to the
IgM+IgD+ PCBCLs took place after their
transition to a germinal center environment, but before the cells lost
IgD expression. Interestingly, PCBCLs no. 5 and 6 were found to express
CD70. These lymphomas may therefore bear a relationship with a recently
described subset of germinal center B cells characterized by expression
of IgD and CD70.24 It was hypothesized that these cells
represent recent immigrants that are in the process of forming a
germinal center. In accordance with this finding, a subset of
IgM+IgD+ germinal center B cells has also been
noticed by others, carrying, on average, 5.8 somatic mutations per VH
gene.5 A minority of these
CD38+IgM+IgD+ B cells harbored more
than 10 mutations in the expressed VH genes. Because the patterns of
somatic mutations in these cells were suggestive of antigen-driven
selection processes, the investigators hypothesized that they might
represent either germinal center founder cells derived from
recirculating IgM+IgD+ memory cells or
centrocytes differentiating into IgM+IgD+
memory B cells.5
The presence of somatic mutations in the VH genes indicates that PCBCLs
may be derived from germinal center cells or their descendants.
Accordingly, in 5 of the 7 PCBCLs studied (no. 2, 3, 4, 5, and 6), the
FRs displayed R/S ratios that are significantly lower than would be
expected if random mutation would have occurred in the absence of
selective forces. The apparant counterselection against R mutations in
the FRs, which are essential for the integrity of the antibody, implies
that expression of proper antigen receptors has been important for cell
survival, at least at some stage(s) of development. However, the fact
that the mutation patterns in these PCBCLs are reminiscent of antigen
selection does not necessarily signify that lymphomagenesis itself is
antigen-driven. In the diffuse large-cell lymphomas no. 1, 2, and 3, no
intraclonal variation was observed. In these lymphomas, the somatic
mutations were most likely introduced before or at the moment of
complete transformation; therefore, it cannot be concluded that these
lymphomas need Ig expression for their survival, let alone antigen
recognition. The fact that the mutation frequency in these cases is
abnormally high may be the result of a prolonged stay in the germinal
center environment, possibly due to early, pretransforming genetic
alterations. The findings within these PCBCLs are in accordance with a
recent report by Gellrich et al,25 who analyzed, using
single-cell PCR, the VH gene of a primary cutaneous immunoblastic
B-cell lymphoma of the leg. It was demonstrated that the VH gene
expressed by this lymphoma harbored 39 nucleotide differences compared
with the most homologous VH gene (DP-54/V3-7). No evidence was obtained for ongoing somatic hypermutation. This subset of large-cell PCBCL, with a high load of somatic mutations without ongoing mutation, resembles noncutaneous diffuse large B-cell lymphomas.19,20 However, our data do not provide an explanation for the difference in
clinical behavior between cutaneous and extracutaneous diffuse large
B-cell lymphomas.
Interestingly, in the PCBCLs no. 4, 5, 6, and 7, intraclonal variation
was found, indicative of ongoing somatic hypermutation. The degree of
intraclonal variation ranged between 0.6 and 3.4 mutations per clone.
In lymphoma no. 7, clonal IgG and IgA gene products were found that
contained the same VDJ rearrangement, indicating that they were isotype
switch variants of the same tumor. It is remarkable that, whereas a
high level of intraclonal variation was observed in the IgG clone (3.4 mutations/clone; Table 4), the IgA clone displayed no significant
intraclonal variation. Still, the consensus sequences of the
IgG-expressing and IgA-expressing clones proved to be exactly the same.
The latter finding may indicate that the Ig heavy chain isotype
switching occurred relatively late after the moment of transformation.
The absence of somatic hypermutation in the IgA subset demonstrates that the ability to mutate may be abolished in the course of disease. This finding may have several explanations. It can be reasoned that the
IgG clones were already functionally heterogeneous and that the isotype
switch to IgA occurred in a nonmutating subclone. Alternatively, the
shut-off of the mutation machinery may somehow have coincided with the
process of isotype switching. It has been demonstrated that, in normal
B cells, heavy chain class switch does not per se terminate somatic
mutation.26 It is possible that additional genetic damage
caused the termination of somatic hypermutation.
The VH mutation patterns of PCBCLs no. 4, 5, and 6 are also suggestive
of clonal selection processes that favor BCR expression. In this subset
of PCBCLs, the somatic mutations found may have been introduced both
before and, contrary to their nonmutating counterparts, after the
moment of complete transformation. It can be assumed that, among the
mutations introduced in the lymphoma cells, there will be those that
give rise to stopcodons or nonfunctional frameshifts. In fact, because
the Ig genes are single-copy genes, they are particulary prone to
inactivation by such mutations. Thus, the finding that actively
mutating lymphomas express Ig is not trivial and implies that the
tumorigenesis of these neoplasms may be BCR-guided. In this respect,
the finding that two of four mutating PCBCLs (no. 5 and 6) contain
elaborate networks of FDCs is noteworthy. Because normal skin does not
harbor FDCs, this suggests that the FDCs may be an essential part of
these neoplasms and possibly have a role in tumorigenesis. This idea is
supported by the strong CD70 expression found on these particular
PCBCLs: CD70 has been described to be a marker for mature B cells that have recently been primed by antigen in vivo.27 On the
other hand, 2 PCBCLs that actively mutate their VH genes are devoid of
FDCs (no. 4 and 7). Assuming that this is not due to not sampling error, this would signify that somatic hypermutation in PCBCLs does not
necessarily depend on the presence of FDCs.
In line with the presumed analogy of the tertiary lymphoid tissue of
skin (skin-associated lymphoid tissue [SALT]) and mucosa (MALT), some
investigators propagate the idea that PCBCLs may be biologically
related to malignant lymphomas of MALT.12,28 On
morphological grounds, this has particulary been suggested for the
immunocytomas of the skin. Although preliminary, our data provide
support for this concept. Of the noncutaneous B-NHLs, ongoing somatic
hypermutation is a characteristic feature of MALT lymphomas29-31 and follicular lymphomas.32-34
MALT lymphomas arise at sites of chronic organ-specific inflammation
caused by autoimmunity or specific infection, eg, Helicobacter
pylori-associated gastritis35 (reviewed in
Wright36). The suggestion of antigen-driven lymphomagenesis was strongly supported by the fact that small-cell gastric MALT lymphomas can be cured with antibiotics that eradicate Helicobacter pylori.37 In this respect, the evolvement of primary
cutaneous immunocytoma in the context of local infection with
Borrelia Burgdorferi 38 is strikingly similar.
Also, for these lymphomas, curative antibiotic treatment has been
documented.39 In the 3 immunocytomas studied here, as well
as in 1 of the diffuse large B-cell lymphomas, we obtained evidence of
ongoing somatic hypermutation in extensively mutated VH genes. In
addition, heavy chain isotype switching, as observed in immunocytoma no
7, has been observed in MALT lymphomas (unpublished
results). The latter phenomenon is also a characteristic capacity of nodal follicular lymphomas.40,41 A potential
relationship between PCBCLs and follicular lymphomas is supported by
the presence of FDCs in the majority of the PCBCLs. Although our data
suggest a relationship between PCBCLs and various forms of noncutaneous B-NHLs, at this moment no definite conclusions are allowed. Additional molecular data on the genetic defects have to be awaited to clarify this relationship.
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ACKNOWLEDGMENT |
The authors thank N.J. Ponne for technical assistance with sequencing
and J.B.G. Mulder and P.J. van Beek for assistance with immunohistochemistry.
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FOOTNOTES |
Submitted March 19, 1998;
accepted July 10, 1998.
Supported by a grant from the Dutch Cancer Society (Grant No. AMC
95-957).
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 C.J.M. van Noesel, MD, PhD, Department of
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Abstract
Introduction