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Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 1864-1872
Clonal Salivary Gland Infiltrates Associated With Myoepithelial
Sialadenitis (Sjögren's Syndrome) Begin as Nonmalignant
Antigen-Selected Expansions
By
David W. Bahler and
Steven H. Swerdlow
From the Division of Hematopathology, Department of Pathology,
University of Pittsburgh School of Medicine, Pittsburgh, PA.
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ABSTRACT |
Myoepithelial sialadenitis (MESA) is the reactive salivary gland
lymphoid infiltrate that occurs in patients with Sjögren's syndrome. Although it is well established that mucosa-associated lymphoid tissue (MALT)-type lymphomas may develop from MESA, the issue
of whether monoclonal B-cell populations in early MESA-associated lesions represent MALT lymphomas or more benign types of expansions has
been very controversial. In addition, it is unknown whether antigen
stimulation plays a role in the development or growth of
MESA-associated clones. To investigate these issues, we have analyzed
the Ig VH genes used by MESA-associated clones in sequential biopsies
obtained from contralateral sites of seven different patients. In three
cases, single clones were identified in the follow-up biopsies that
were distinct from the single clones identified in the initial
specimens, whereas in three other cases, the same clone was identified
in both the initial and subsequent specimens. In the remaining case,
two clones were identified in the second biopsy specimen, one of which
was distinct from the initial clone. Of the 11 distinct clones
identified in the 14 specimens that were analyzed, 8 were derived from
a V1-69 VH gene segment, whereas the other 3 were derived from a V3-7
VH gene segment. In addition, the MESA clones also showed conserved
amino acids sequence motifs in their third complementarity-determining
regions (CDR3), some of which were encoded by N nucleotides. The marked
VH gene restriction along with the similar CDR3 sequences suggests that
MESA-associated clones even from different patients may bind the same
or similar antigens and are selected for clonal expansion on that
basis. The high rates of ongoing VH gene mutation observed in some of the cases futher suggest that the growth of early MESA clones is still
dependent on antigen stimulation. In addition, our finding that
different biopsies from the same patient may contain distinct clones
indicates that some MESA-associated clones have not yet evolved to
malignant lymphomas.
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INTRODUCTION |
MOST PRIMARY SALIVARY gland lymphomas are
of mucosa associated lymphoid tissue (MALT) type and are thought to
arise from a reactive infiltrate termed myoepithelial sialadenitis
(MESA) that is also known as benign lymphoepithelial
lesion.1-3 MESA is found in patient's with
Sjögren's syndrome and is responsible for the salivary gland
dysfunction that occurs in this disease but can also be seen in
patients who may not have Sjögren's syndrome. The transition
from reactive MESA to monoclonal lymphoma is not well understood,
although there has been speculation that chronic stimulation by
exogenous or autoantigens is playing an important role by driving the
proliferation of specific B cells and increasing the frequency of their
transformation.4-8 Reports that the proportion of B cells
expressing the 17.109 and G6 idiotypes are increased in salivary gland
biopsies from patients with Sjögren's syndrome compared with
controls suggest that the B-cell infiltration early on represents a
selected population.5-7 Antigen selection of specific B
cells for transformation was also suggested by our earlier study of
salivary gland MALT lymphomas, in which 3 of the 5 cases examined used
the 51p1 VH and Humkv325 VL genes that encode the G6 and 17.109 idiotypes, respectively.8 A role for antigen stimulation in
the growth of MALT lymphomas has also been proposed for those that
occur in the stomach that are thought to develop out of
Helicobacter pylori-associated gastritis.9 Remarkably, several studies have now shown that the majority of low-grade gastric MALT lymphomas may be curable using only antibiotics that eliminate the H pylori infection.10,11
It is well recognized that early salivary gland MALT lymphomas can be
very difficult to distinguish from reactive MESA, which is also
invariably present.1 Histologic criteria to make this distinction are controversial and have evolved with the recognition that MALT-type lymphomas represent a distinct type of extranodal lymphoma.1,12,13 The significance of finding monoclonal
B-cell populations by polymerase chain reaction (PCR) or Southern blot analysis is also unclear, because they have been detected in
MESA-associated salivary gland lesions that histologically appear
benign.13,14 Although it is possible these clones represent
very early lymphomas, as some have argued,15,16 clones in
histologically benign MESA or early low-grade MALT lymphomas often
behave clinically in a very indolent fashion and can remain localized
to the salivary glands for years without evidence of other
spread.13,17 Moreover, it is well recognized that patients
with MESA and Sjögren's syndrome often have monoclonal
paraproteins or cryoglobulins that are thought to reflect a stable
nonmalignant lymphoproliferative disorder.18
To further evaluate the malignant potential and evolution of B-cell
clones in MESA-associated salivary gland lesions, we studied the clonal
VH genes in successive biopsies from seven patients that had been
obtained from contralateral sites. Sequence analysis of VH genes can
provide considerable information regarding how different clones may be
related and whether antigen selection may be playing a role in clonal
evolution.19-21 Although both MESA and MALT lymphomas
frequently show bilateral involvement of salivary glands, the
relationship, if any, among clones from such lesions has not been
extensively studied.17,22 Finding that different biopsies
of MESA or low-grade MALT lymphoma from the same patient can contain
different clones supports a model of lymphomagenesis in which the
emergence of monoclonality in the salivary gland precedes the onset of
malignant lymphoma. Moreover, the restricted use of VH gene segments
and CDR3 sequences we found strongly suggest that the B-cell clones
associated with MESA lesions in the salivary gland arise through the
process of antigenic selection.
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MATERIALS AND METHODS |
Patient material and DNA isolation.
The 16 biopsy specimens were obtained from patients seen at the
University of Pittsburgh Medical Center and satisfied histologic criteria for MESA, MESA with halos of monocytoid cells, or low-grade MALT lymphoma as described.13 DNA was isolated from the
three fresh tissue biopsy specimens (HA #1 and #2, OR #1) as
described.8 For the remaining 13 biopsies, for which only
paraffin-embedded tissue was available, DNA was isolated using a
different method, as described.23
Amplification of rearranged VH genes.
DNA extracted from the biopsy specimens was initially amplified as
described24 for 37 cycles with a consensus primer specific for the 3 end of VH gene framework three (FW3) regions and the consensus JH1 primer (Table 1).
Approximately one third of the resultant products were electrophoresed
in 8% acrylamide gels, and prominent bands indicative of B-cell clones
were identified by visual inspection after staining with ethidium
bromide. Rearranged VH genes were further amplified from the biopsy
specimens in which clonal bands were identified using a variety of
5 primers specific for framework one (FW1) regions (Table 1) in
conjunction with a consensus JH primer in individual reactions
containing a single FW1-JH primer combination. After 35 cycles of
amplification, 0.5 µL of the resultant products was reamplified with
internal FW1 or JH primer for 15 cycles. Typically, the internal JH2
primer would be substituted for JH1, although in some cases in which the JH2 primer did not appear to bind because products were not generated, internal FW1 primers were used in the secondary reactions. This nested approach was necessary with the paraffin-extracted DNA
templates to generate easily identifiable VH bands after
electrophoresis in 1.5% agarose and ethidium bromide staining or to
obtain sufficient quantities of DNA for subsequent cloning.
Cloning and sequencing of PCR products.
After electrophoresis in 8% acrylamide, clonal FW3-JH1 bands
were isolated from gels and incubated for 24-72 hours in 500 µL of TE
at 37°C. After centrifugation, the TE solution was precipitated with sodium acetate, and the resulted DNA pellet was resuspended in
H2O. Appropriately sized VHFW1-JH PCR products were
isolated from 1.5% low melt agarose gels and further purified using
Wizard DNA preps (Promega, Madison, WI). Approximately one seventh of the purified DNAs were cloned using the PCR-Script kit (Stratagene, LaJolla, CA). Plasmid DNA was isolated from overnight 1.5-mL cultures of randomly selected colonies using Wizard mini-preps (Promega). Diodeoxy sequencing was performed with Sequenase (US Biochemical, Cleveland, OH) following the manufacturer's protocol using
approximately one fourth of the isolated plasmid DNA. Clones were
sequenced in both directions using the M13 forward and reverse primers. In several cases, the small FW3-JH1 products were directly sequenced without cloning. Single-stranded templates for these sequencing reactions were generated by subjecting one third of the purified DNAs
to 20 cycles of amplification with either the FW3 or JH1 primer. After
precipitation with ammonium acetate, sequencing was performed as
described above using one half of the precipitated single-stranded DNA
with the primer not used to generate the templates.
Analysis of VH gene mutations.
Expected numbers of replacement (R) and silent (S) mutations in the
first and second compementarity-determining regions (CDR) and VH
framework regions (FWR) were calculated as described by Chang and
Casali.25 Any shared mutations found in several clonally related VH sequences were counted only once. The calculated mutation values were rounded to the nearest integer.
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RESULTS |
Identification of B-cell clones.
Two contralateral biopsy specimens of MESA-associated lesions
from eight different patients were analyzed by a standard PCR technique
for detecting clonal B cells. In seven of the eight cases, both of the
contralateral biopsies, which are further described in
Table 2, contained clones. The biopsies
from one case showed only polyclonal B cells and were not further
studied. With the exception of entries for patient HA, most of the
other information in Table 2 can be found in our earlier study (see
Table 7 in Quintana et al13) along with other clinical data
for these patients. Biopsies with material available for analysis in
this study were largely from parotid glands and the time intervals
between the first and second biopsies ranged from several months to
more than 10 years. Histologically, most of the biopsies met criteria
considered to be diagnostic of low-grade MALT lymphoma by showing
confluent areas of pale monocytoid cells
(Fig 1). However, several of the biopsies
showed only MESA with small nonconfluent halos of monocytoid cells
around the lymphoepithelial lesions, which has been proposed to
represent a precursor lesion or the earliest histologic sign of
lymphoma. In one case (MA), both of the biopsies that were analyzed
histologically showed only reactive MESA.
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| Fig 1.
Histologic features of the low grade salivary gland MALT
lymphoma from patient JA. Note the halos of pale monocytoid cells around lymphoepithelial lesions on the right that become more confluent
particularly on the left.
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Representative clonal bands after electrophoresis and ethidium bromide
staining of FW3-JH PCR products are shown in Fig 2 for
three of the cases. Observe that for case PO, single clonal bands of
the same size are present in both biopsy specimens, whereas case LE
shows single clonal bands in both biopsies that have different sizes.
In case BA, at least two prominent monoclonal bands can be seen in the
second specimen, with one being the same size as the single band in the
first specimen. The clonal bands were purified and sequenced, which in
all cases yielded a unique in frame clonal CDR3 sequence
(Fig 3). For three of the cases (JA, PO,
and HA), the same single clone was present in both biopsy specimens,
because each had identical or nearly identical CDR3 sequences. However, for three other cases (MA, LE, and OR), the single clones identified in
the two biopsies were different and had distinct CDR3s due to the use
of different D segments and N nucleotides. In case BA, two different
potentially functional clones were identified in the second biopsy, one
of which was the same as the initial clone.
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| Fig 2.
PCR clonality analysis. Shown are representative products
produced by amplifying paraffin-extracted DNAs from three cases with
the consensus FW3 and JH1 primers after electrophoresis and ethidium
bromide staining. Single clonal bands are seen in all of these
specimens except in the second biopsy from patient BA, which shows
multiple clonal bands. Molecular weight markers are shown in the left
most lane. Each lower rung of the polyclonal background ladders
represents 3 nucleotides or one amino acid.
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| Fig 3.
CDR3 nucleotide sequences. The clonal CDR3 sequences in
the second biopsies are compared with those identified in the initial biopsies. Whether the sequences were derived from directly sequencing FW3-JH1 PCR products or from sequence analysis of individual clones of
FW3-JH1 products is indicated in parentheses along with the number of
related clones to total clones analyzed. N nucleodies are shown in
lower case, whereas D and J segments are shown as boldface capitols (J
segments left most). Nucleotide differences from the proposed germline
D and J segments are underlined. (The names of the D and J segments are
given in Table 3.)
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Characterization of clonal VH genes.
To further study these clones, rearranged VH genes were amplified from
the specimens using a variety of FW1-JH primer combinations, and the
products from some of these reactions were cloned and sequenced.
Complete or nearly complete VH gene nucleotide sequence information was
obtained for all of the B-cell clones identified previously with the
FW3-JH1 primers, because the VH genes had the same CDR3 sequences. The
two VH genes from biopsies HA#1 and OR#2 were described in an earlier
study and were amplified using VH leader primers.8
Comparing the VH sequences to those in the VBASE and GeneBank
directories indicated that the 11 clonal VH genes we had identified
with distinct CDR3s were most closely related to germline VH gene
segments from only two loci, V1-69 and V3-7
(Fig 4 and
Table 3). Of the eight distinct V1-69
genes, five are most homologous to the 51p1 gene and three are most
homologous to the closely related allelic variant 7M27, which only
differs from 51p1 by one nucleotide in FW3.26 In addition
to the frequent use of specific VH gene segments, comparing the clonal
CDR3 sequences to known germline elements also showed evidence of
restricted use of J and D segments. For the eight distinct V1-69 genes,
seven appear to use a J4 segment and five of these use D segments from the DXP family, D21-9 (4 genes) and DXP 1 (1 gene), in the same second reading frame. For the three V3-7 genes, all use a J3 segment and D segments from the DXP family, D21-9 (2 genes), and DXP 1 (1 gene) also in the second reading frame. Some of the CDR3s also show
evidence of shared amino acid residues encoded by N-nucleotides at the
VH-D junction. All three of the V3-7 CDR3s start with glycine and
aspartate residues (GD single letter code,) whereas four of eight V1-69
genes have glutamate (E single letter code) as the first amino acid
(Fig 4).
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| Fig 4.
Deduced amino acid sequences of the clonal VH genes.
Differences from the germline gene segments are shown as upper case
letters or as underlined letters in the CDR3s. The locations of silent mutations in the nucleic acid sequences are indicated with lower case
letters. Only mutations that were present in all of the PCR clones are
shown, ie, the common mutations. The VH genes derived from 2m27 (JA-1,
BA-1, and OR-1) do not have an E to K mutation in the eighth FW3
position, because this represents the single nucleotide difference
between the 2m27 and 51p1 VH gene segments. Sequence gaps in the CDR3s
identifies the J segments start. These sequence data are available from
Genebank under accession nos. AF038442-AF038450 and U79591-U79592.
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Mutation analysis.
Numerous single nucleotide differences or mutations between the VH
genes and the proposed germline sequences are evident in all of the
clones (Fig 4 and Table 3). Some of these mutations were seen in all of
the clonally related sequences analyzed from a given specimen, the
common mutations, whereas others were only present in some of the
clonal VH sequences, the noncommon mutations. In the four cases in
which the initial clone was also identified in the second contralateral
biopsy (PO, JA, HA, and BA), unique common mutations from germline were
present in both clones, indicating that each site contained a distinct
subclone. The number of noncommon mutations identified in many of the
clonal VH genes was substantial and in some of the clones greater than
the number of common mutations. The locations and type of noncommon
mutations in individual clones for the two VH genes with the greatest
number of noncommon mutations are depicted in
Fig 5. Observe that many of the noncommon
mutations are shared between different VH gene clones, indicating their sequential development or stepwise evolution. The noncommon mutations in these two VH genes are also tabulated in
Table 4 and compared with the expected
distributions of R and S mutations, assuming the mutations occurred by
chance alone. As can be appreciated, the CDRs as well as FWRs have
fewer R mutations and lower R/S ratios than expected, which suggests
that there is selection against R mutations in both of these regions.
Findings similar to these were also observed in the other clonal MESA
VH genes that showed significant intraclonal heterogeneity.
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| Fig 5.
Diagramatic representation of the noncommon mutations in
the different VH clones from patients JA (A) and BA (B), the two VH
genes with the greatest number of noncommon mutations. Silent (S)
mutations are shown as open boxes, whereas replacement (R) mutations
are indicated by solid boxes. The four codons that contained both S and
R mutations are indicated by partially solid boxes.
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DISCUSSION |
The significance of finding monoclonal B cells in MESA-associated
infiltrates by PCR or Southern blot analysis has been controversial. Fishleder et al14 as well as Knowles et al27
suggested that these clones may represent nonmalignant expansions,
because they can be frequently found in lesions that histologically
appear benign and often remain localized for prolonged periods of time. However, these studies were completed several years before MALT lymphomas were generally recognized as distinct entities and the histologic features of their cases were not described in detail. More
recently, Isaacson and others16,22 have argued that
MESA-associated monoclonal B-cell populations represent
malignant lymphomas, because they are usually associated with
identifiable histologic features (halos or confluent areas of
monocytoid B cells around lymphoepithelial lesions) and their
propensity to remain localized simply reflects the unusually indolent
behavior that is characteristic of low-grade MALT type lymphomas.
To help clarify the nature of clonal MESA-associated lymphoid
infiltrates, we analyzed VH clones in sequential contralateral biopsies
from seven different patients to determine how they may be related to
one another. It is important to emphasize that the PCR technique we
used to initially identify the clones is very specific even with DNA
extracted from paraffin-embedded tissue that is often of poor quality
because the amplification products are relatively small consisting
mostly of CDR3 regions.28,29 Although false-positive clonal
bands can occasionally be detected with this method from samples
containing very few B cells when the DNA is of poor quality, this was
not a problem even from our paraffin-extracted templates, because all
demonstrated abundant B cells as shown by the prominent polyclonal
signals that were seen along with the clonal bands. In terms of
sensitivity, previous dilutional studies with polyclonal DNA indicated
that a clone making up approximately 2% of a B-cell population
represented our lower limit of detection, which is close to the
detection limit of very sensitive Southern blot analysis. It is likely, therefore, that all of the clones we identified by PCR could also have
been detected by Southern blot analysis if fresh tissue had been
available. Indeed, for the three fresh tissue biopsies that were
analyzed, Southern blot and our PCR clonality test gave concordant results.
In three of the seven cases that were studied, a single VH clone was
identified in the follow-up biopsy that appeared to be clonally
unrelated to the single VH clone identified in the initial biopsy,
because it had a distinct CDR3 sequence with different D segments and N
nucleotides. Because all of the clonal VH sequences we identified were
in frame and free of stop codons, each could correspond to a distinct
B-cell clone. Thus, finding distinct VH clones in the contralateral
biopsies cannot be readily explained by differential amplification at
each site of the productive and nonproductive allele from the same B
cell. Similarly, in the one case in which two VH clones were identified
in the follow-up biopsy, one of which was clonally distinct from the
initial clone, each appears to correspond to a separate B-cell clone as
opposed to the productive and nonproductive allele of the same clone.
The possibility that our finding different clones resulted from a specimen labeling error was formally ruled out by PCR analysis of the
androgen receptor gene, which gave a pattern of bands that was unique
for each patient because of the variable number of CAG
repeats30 but identical between paired specimens (not
shown). Finding distinct B-cell clones in different biopsy specimens
from the same patient strongly argues that monoclonal MESA-associated expansions are not necessarily malignant lymphomas. Moreover, in two of
the patients with different clones in the second biopsies, the MESA
lesions were also not histologically diagnostic of lymphoma.
Evidence that different B-cell clones may be present in sequential
biopsies of MESA-related lymphoid infiltrates was also reported by
Fishleder et al.14 In that study, Southern blot analysis
showed that the salivary gland biopsies obtained 2 years apart from one
patient contained different clonal heavy chain and light chain
rearrangements. However, because the Ig gene somatic hypermutation
mechanism can be active in MALT lymphomas,8,31 it was still
possible that the different rearrangements did not represent separate
clones but resulted from the creation of new restriction endonuclease
sites in the same clone, similar to what has been described for
follicular lymphomas, which also mutate their Ig genes during clonal
expansion.32 A recent study by Diss et al22
that used a PCR clonality technique similar to ours found all of the
clonal bands to be of similar size in the sequential salivary gland
biopsies from the six patients that were analyzed. However, in that
study, the PCR bands were not sequenced, so it is possible that some
may have been different clones similar to two of our cases in which the
different clones had PCR bands that were the same sizes. Our greater
success in identifying different clones may also be related to all of
our sequential biopsies being from contralateral locations and to our
inclusion of early MESA-associated lesions that histologically were not
diagnostic of MALT lymphoma.
This study further establishes that MESA-associated clones, even from
different patients, are not completely unrelated but often express VH
genes that are remarkably similar to one another. One aspect of the VH
gene similarity results from biased use of certain VH gene segments.
Recent estimates indicated that there are approximately 50 different
functional VH gene segments in the human genome.33
Therefore, finding that the 11 MESA clones with distinct CDR3s
described in this study use either V1-69 (8 clones) or V3-7 (3 clones)
represents a highly nonrandom VH gene repertoire. Besides the biased
use of VH gene segments, different MESA clones also contained similar
amino acid sequence motifs in their CDR3s. Some of this CDR3 similarity
can be explained by the frequent use of D-segments from the DXP1 family
in the second reading frame in 8 of 11 clones, the use of J4 segments by seven of eight V1-69-derived clones, and the use of J3 segments by
three of three V3-7 genes. In addition, identical amino acids were also
noted at the 5 end of many CDR3s (GDY in 3 of 3 V3-7 genes and E
in 4 of 8 V1-69 genes), which appear to correspond to residues encoded
by N nucleotides. These findings, along with the VH gene restriction
noted above, strongly suggest that MESA-associated clones represent a
highly restricted B-cell subset that has been selected for expansion
through their Ig molecules.
The VH gene similarities between the two different clones identified in
case MA are so striking that a cursory analysis might suggest they were
clonally related to one another. Both used the same VH and JH segments
and also had CDR3s that were the same sizes that only differed by 3 of
15 aminoacids, two of which were conservative glycine to serine (G to
S) substitutions. However, it is unlikely that the G to S substitutions
that were located in the D segments resulted from somatic mutations in
the initial clone, because there was near perfect homology with
substantial portions of the two different but related D21-9 and DXP'1
D segments that were proposed to be used with matches at 21 of 21 and
18 of 19 nucleotides, respectively, and the G to S substitutions represent germline areas of these D segments. In addition, no mutations
were identified in any of the 12 individual PCR clones that were
analyzed that corresponded to the 8 nucleotide differences in the CDR3
sequences between these genes. Further evidence that the two MA clones
are indeed independent comes from consideration of the V3-7
MESA-associated clone we identified in our previous study that also had
a CDR3 that was remarkably similar to the initial MA clone differing by
only two D to E and H to I substitutions excluding its one additional
amino acid of length.8 Moreover, the additional V3-7 MESA
clone that was identified in this study also has a CDR3 sequence that
is remarkably similar to those used by the MA clones. The very high
degree of VH and CDR3 sequence similarity seen between different
MESA-associated clones, especially those using V3-7, suggests that some
may bind the same epitope or antigen.
Although the antigen specificity of the MESA clones is unknown,
antibodies encoded by V1-69 and V3-7 have been described that display
reactivity toward a variety of autoantigens, including IgG and
DNA.34,35 Further support that the MESA clones may have
anti-IgG or rheumatoid factor activity comes from their CDR3 sequences,
because rheumatoid factors that use V1-69 or V3-7 typically have highly
distinctive CDR3s compared with randomly chosen V1-69- or
V3-7-derived VH genes.34 Specifically, like the MESA
clones, rheumatoid factors derived from V3-7 usually have CDR3s that
start with the amino acids glycine-aspartate-tyrosine (GDY), frequently use the D21-9 and J3 gene segments, and are typically 14 to 15 amino
acids in length. In addition, the MESA clones using V1-69 also resemble
the V1-69 rheumatoid factors in having CDR3s that often start with
glutamate (E), frequently use the J4 segment, and use
asparagine-proline (NP) residues at several D-J junctions. The
possibility that MESA-associated clones frequently have rheumatoid factor activity is also consistent with the increased salivary gland
production of rheumatoid factors reported in patients with Sjögren's syndrome.36
It is interesting that V1-69 VH genes are also frequently expressed by
chronic lymphocytic leukemia (CLL), a low-grade malignancy of CD5
positive B cells, being found in approximately 20% of
cases.21 This raises the possibility that CLL and salivary
gland MALT lymphomas may be related in terms of pathogenesis and
derived from the same selected B-cell subset. However, from analysis of
these few cases, the restricted types of CDR3 sequences frequently used
by V1-69-derived MESA and CLL VH genes appear to be quite different.
For example, J6, which was not used by any of our eight MESA V1-69
sequences, was used by 12 of 26 CLL V1-69 genes described in a recent
study by Johnson et al,37 whereas D21-9, identified in five
of our eight MESA V1-69 sequences, was used by only 1 of these 26 CLL sequences. Because residues encoded by CDR3 frequently affect antigen
binding, these observations suggest that, although CLL and
MESA-associated clones may be highly selected populations, the V1-69
Igs from each will have different antigen specificities.
Antigen selection of the MESA clones is also supported by analyses of
the numerous mutations that were found the VH genes. It is well
established that VH genes expressed by MALT lymphomas are typically
mutated from germline and often show considerable intraclonal variation
indicative of ongoing Ig gene mutation.8,31 Replacement (R)
mutations in the CDRs, which encode residues that form the conventional
antigen binding site, can be selected for (positively selected) if they
increase binding affinity or selected against (negative selection) if
they lead to lower antigen binding affinity.38 In
evaluating mutations for evidence of clonal selection, mutations that
are known to have been acquired as the clone expands are potentially
the most informative. These correspond to mutations that are present in
only some of the clonally related VH sequences, the noncommon
mutations, as opposed to mutations present in all of the VH sequences,
the common mutations, which could have occurred before expansion or in
a B-cell located in a different location.19 Analyses of
noncommon mutations was facilitated in this study by finding four MESA
VH genes with very high ongoing mutation rates of greater than 2%,
which is considerably higher than what has been previously reported for
MALT lymphomas.8,31,39 Instead of high numbers of noncommon
R mutations in the CDRs indicative of positive selection, the
MESA-associated clonal VH genes with significant intraclonal
heterogeneity all contained fewer noncommon R mutations in the CDRs
than expected by chance, indicating there is negative selection or
selection against R mutation. Moreover, the ratios of R to S mutations
in the CDRs were all less than 1.5, whereas ratios of R to S mutations
in the V1-69 and V3-7 genes expected without selection should be 3.6:1
and 3.9:1, respectively. Negative selection suggests that clonal
expansion is dependent on the preservation of residues in the CDRs that
are important for antigen binding and has been previously described for
rheumatoid factors that use the 51p1 gene.40 Negative
selection of R mutations also appears to be operating in the FWRs,
because they also have fewer noncommon R mutations and lower R/S ratios
than expected by chance. Because the maintenance of Ig function
requires preservation of many FWR amino residues, this further suggests
that functional Ig is important for clonal proliferation.38
Although our earlier study also provided evidence of negative selection
of R mutations in the CDR and FWR of several salivary gland MALT
lymphoma VH genes,8 that analysis was limited to mostly
common mutations that may be less informative, as described
above.
The reason for the variability in ongoing mutation rates among these
clonal MESA-associated VH genes, which ranged from 0% to 4.0%, is
unclear. However, one possibility is that those clones with low or
nondetectable ongoing mutation are less dependent on antigen
stimulation, because they have acquired additional oncogenic events
that could substitute for an Ig-mediated growth signal. This idea is
also consistent with our finding that the average ongoing mutation rate
in clonal VH genes from more benign MESA-associated lesions twice the
rate found in clones from lesions that histologically were diagnostic
of MALT lymphomas (1.8 v .74). With the high rates of ongoing
mutation in some of the clonal VH genes, it may also be significant
that no stop codons were identified in any of the 86 total individual
VH PCR clones or 120 noncommon mutations that were analyzed. This
further supports the concept that expression of functional Ig molecules
is important for the survival and proliferation of early
MESA-associated clones.
In summary, the highly restricted VH gene repertoire and ongoing
mutation analysis indicates that Ig is playing an important role in the
development and subsequent expansion of MESA-associated clones. The
most straightforward model to propose is that B cells with the
appropriate VH structures are selected for expansion through surface Ig
stimulation by binding a common antigen
(Fig 6). At some point, a genetic event
probably occurs in one of the expanding clones that gives it a growth
advantage over the others, because it is unlikely antigen stimulation
by itself could generate a clonal population that would be large enough
to be detected by PCR analysis. However, the development of clonality
in MESA may be aided by immune system dysregulation, which is part of an autoimmune disease process. Because patients with MESA typically have involvement of multiple glands, independent clones can sometimes develop at different sites in the same patient, as we have shown. It is
unlikely that clones that first become detectable by PCR are malignant,
because no patients have been described with disseminated lymphomas in
different organs that are clonally distinct.32,39,41 Moreover, it is well established that not all monoclonal lymphoid proliferations behave in a malignant fashion.42-44 We
propose, therefore, that additional oncogenic events after the
emergence of monoclonality are usually required before MESA clones
become malignant lymphomas capable of dissemination and growth outside of the salivary gland. It is possible that trisomy 3, a cytogenetic abnormality frequently found in MALT lymphomas,45
represents such an event or mutations in the P53 gene that have been
associated with histologic progression of MALT lymphomas.46
Although the exact point at which lymphoma develops remains to be more
precisely defined, our data suggest that the early nonmalignant clones
are more likely to be found in MESA-associated lesions that are
histologically more benign in appearance. From a practical standpoint,
therefore, patients with a histologically benign or borderline MESA
lesion that contains a clonal population may not require local
antineoplastic therapies, as has been suggested,15 given
the potential morbidity. Moreover, the rationale for local therapy
might also be questioned in this situation, because additional clones
may be present in a contralateral salivary gland.
View larger version (10K):
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| Fig 6.
Schematic model of MALT lymphomagenesis in the salivary
gland. Clones detectable by PCR or Southern blot analysis develop in
reactive MESA as a result of direct stimulation by a limited number of
antigens and the occurrence of a premalignant oncogenic event.
Initially, the clones are still dependent on antigen stimulation for
growth and remain localized, although some may be capable of spreading
to other salivary glands that are also involved by MESA. At some point,
which may be many years after a clone first appears, additional
oncogenic events occur that result in the clones becoming malignant
lymphomas capable of widespread dissemination and growth in other
organs.
|
|
| |
FOOTNOTES |
Submitted November 4, 1997;
accepted December 16, 1997.
Supported by the Pathology Education and Research Foundation
(University of Pittsburgh) and Grant No. IRG-58-34 from the American Cancer Society.
Address reprint requests to David W. Bahler, MD, PhD, Department of
Pathology, Presbyterian University Hospital C604, 200 Lothrop St,
Pittsburgh, PA 15213.
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.
| |
ACKNOWLEDGMENT |
The authors thank John Miklos for technical assistance and Diana
Winters for secretarial assistance.
| |
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Abstract
Introduction
Abstract