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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4292-4299
Analysis of VH Genes in Follicular and Diffuse Lymphoma
Shows Ongoing Somatic Mutation and Multiple Isotype Transcripts in
Early Disease With Changes During Disease Progression
By
Christian H. Ottensmeier,
Andrew R. Thompsett,
Delin Zhu,
Bridget S. Wilkins,
John W. Sweetenham, and
Freda K. Stevenson
From the Molecular Immunology Group, Tenovus Laboratory, Southampton;
and the Departments of Pathology and Medical Oncology, Southampton
University Hospitals, Southampton, UK.
 |
ABSTRACT |
Investigations of VH gene mutational patterns in B-cell
tumors are often performed at an arbitrary time point of disease. To
assess the effects of disease progression, tumor-derived VH genes have been monitored from presentation through treatment and
relapse in one patient with follicle center lymphoma (FCL), and two
patients with primary diffuse large B-cell lymphoma (DLCL). The patient
with FCL and one patient with DLCL both achieved clinical remission,
although this was only partial in the FCL. However, both subsequently
relapsed, and the second patient with DLCL was refractory to
radiotherapy and chemotherapy. In each case, the tumor-derived
VH sequence was identified, and the CDR3 "clonal signature" was used to track tumor cell sequences in subsequent biopsies. All cases showed somatic mutations, with intraclonal heterogeneity evident at presentation, and some sequences were aberrant. The VH sequences of the DLCL which responded to
treatment became homogeneous at relapse. The sequences of both the FCL
and the refractory DLCL remained heterogeneous. In all cases,
transcripts of multiple Ig isotypes could be identified, and there was
immunophenotypic evidence for expression of several Ig isotypes. The
case of refractory DLCL had identifiable transcripts from IgM, IgD,
IgA, IgG, and IgE, but appeared to lose the ability to produce
alternative isotype transcripts and protein at the late stage of
disease. These cases indicate that VH gene analysis can be
used to probe tumor cell behavior in cases of lymphoma and that
perturbations caused by therapy and disease progression can occur.
| |
INTRODUCTION |
THE ABILITY TO identify and sequence Ig
variable region genes is providing information relevant for
understanding the behavior of B-cell tumors. Recombination of the three
genetic components of the heavy chain to create the
VH-DH-JH transcriptional unit is a
unique feature of B cells. Subsequent events of
VL-JL recombination, somatic mutation, isotype
switching, and antigen selection all leave evidence in the V-gene
sequences of the stage of differentiation reached by the B cell of
origin. This clonal history is maintained in the neoplastic B cell and
is providing insight into B-cell differentiation and tumor
development.1,2
VH gene analysis has shown that chronic lymphocytic
leukemia (CLL) may be heterogeneous, with one subset derived from naive B cells with unmutated sequences, and another from cells that have
acquired somatic mutations.3 Because the mutational
machinery is likely to be activated in the germinal center of the
lymphoid follicle,4 this indicates that, in some cases, the
cell of origin may have traversed this site. Similar analysis has shown that VH genes from cases of follicle center lymphoma (FCL)
have undergone somatic mutation and continue to accumulate mutations after malignant transformation.5,6 This gives rise to
VH sequences with a clear, common "clonal signature"
in the recombination site (CDR3), but with nucleotide differences
between the sequences generating intraclonal
heterogeneity.6 Cases of endemic or sporadic Burkitt's
lymphoma have a similar pattern.7 In contrast, cases of
putatively more mature lymphomas such as splenic lymphoma with villous
lymphocytes8 or multiple myeloma,9,10 although somatically mutated, show no intraclonal heterogeneity, indicating that
the final neoplastic event may have occurred at a postfollicular stage.10
Primary diffuse large B-cell lymphomas (DLCL) are aggressive lymphomas
and represent 30% to 40% of B-cell malignancies.11 Typically they present with rapid growth at the site of involvement and
require combination chemotherapy. Primary DLCL can be distinguished from cases of FCL which have undergone blastic transformation, in
having no preceding low-grade phase or accompanying histological features of low-grade disease. Typically, DLCL are composed of sheets
of centroblastic or immunoblastic cells.11 In a study of
VH genes in tumors from 17 patients with primary DLCL, the most notable finding was of biased VH gene usage, with the
V4-34 gene involved in 11 of 17 cases.12
Significant levels of somatic mutation were present, but no intraclonal
variations. This suggested that DLCL was distinct from FCL
in that mutations were not accumulating following neoplastic
transformation. However, it was pointed out in the study that
these patients were mainly at a late stage of relapse after
treatment.12
After somatic mutation, a normal B cell either undergoes selection by
antigen or dies by apoptosis.13 It remains possible that
antigen may play a role in driving growth of neoplastic B cells, but
tumor cells have various devices to escape apoptotic death.6 The question of whether a tumor cell can also
differentiate further after neoplastic transformation, which is
suggested in tumors such as lymphoplasmacytoid lymphoma by
morphology,11 is being investigated at the genetic level.
Phenotypic analysis shows that a few tumors can undergo isotype
switch.14 Analysis of transcripts defines this ability more
clearly and has shown that, in CLL, alternative isotype production may
be quite common.15-19
Deductions concerning the cell of origin are usually made from
VH gene analysis of a single biopsy specimen. However,
we20 and others5 have shown that the genetic
profile of lymphoma can change with disease progression. This has been
observed in FCL which, after intensive chemotherapy, can show a
narrowing of intraclonal heterogeneity toward
homogeneity.20 One possibility is that this reflects a
second event in one cell which allows escape from
chemotherapy.21 To analyze this more closely, we have
studied three cases of lymphoma from presentation through disease
progression. We have shown that changes in VH gene
mutational pattern may occur during treatment and that intraclonal
variation is present in DLCL in early disease. We have also found
multiple alternative isotype transcripts in all three cases, and have
suggestive evidence that the range of isotypes may diminish with
progression.
| |
MATERIALS AND METHODS |
Patients and histological assessment.
Cases were chosen randomly based on the availability of tissue from the
time of diagnosis and at least one further sample at a relapse or
disease progression. Clinical information was obtained from case
records in the CRC Wessex Oncology Unit (Southampton, UK),
and the course of disease in the three patients (BA, EJ, and BR) is
summarized in Table 1. The slides of all
samples were reassessed by an experienced lymphoma pathologist
(B.S.W.). Archival biopsy material was available either as frozen
tissue stored in liquid nitrogen (F biopsies), or as paraffin-embedded
tissue (P biopsies). Stage at presentation and treatment outcome was
assessed using standard criteria (clinical examination, chest
radiograph, ultrasound/computer tomography of the abdomen ± chest and bone marrow [BM] aspirate + trephine, full blood count
[FBC], and serum biochemistry). Patient BA with
marrow-positive FCL was treated with chlorambucil and achieved a
partial remission with one residual submandibular node. A frozen tissue
biopsy (BA-1F) was available from presentation, and a paraffin-embedded
biopsy (BA-2P) from a second site at relapse. Patient EJ with DLCL also
only achieved partial responses to two standard regimens of combination
chemotherapy.22,23 Frozen tissue biopsy specimens were
obtained at presentation (EJ-1F), and from a tonsil (EJ-2F) at first
progression. A third frozen tonsillar biopsy (EJ-3F) was available
after second relapse. Patient BR with testicular DLCL
achieved complete clinical remission but relapsed after 14 months. In
this case, a paraffin-embedded biopsy (BR-1P) only was available at
presentation, but a frozen biopsy (BR-2F) from a cervical lymphnode was
obtained at relapse.24
To confirm the findings in the cases of DLCL a group of seven more
presentation biopsy specimens from primary nodal B-DLCL have since been
studied.
Immunohistochemistry.
For analysis of Ig light- and heavy-chain expression, cryostat sections
were cut from frozen samples and stained using an established indirect
immunoperoxidase method.25 The primary monoclonal
antibodies (MoAbs) used were anti-IgD (DAKO, High Wycombe, UK), IgM,
IgG, and IgA (kind gift from Dr M. Glennie, Tenovus Laboratory). Negative controls were performed by omitting
the primary antibody.
Paraffin section immunohistochemistry was also performed to assess the
expression of B-cell-associated (CD20, CD79a) and T-cell-associated (CD3, CD45RO) antigens (all from DAKO) plus Ki67 antigen
as an estimator of the proliferating fraction of tumor cells. An
established streptavidin-biotin complex immunoperoxidase method was
used with appropriate antigen retrieval.26
Preparation of cDNA from frozen biopsy samples.
Frozen tissue was transferred into the cold chamber of the cryostat and
embedded in OCT compound (R. Lamb, London, UK). Three to
four 5-µm sections were cut and transferred to sterile Eppendorf vials. Extraction of RNA was performed in a laminar flow
hood using 800 µL RNAzol (Cinna Biotecx Labs, Inc, Houston, TX).
Twenty microliters of solution was reverse transcribed using an
oligo-d(T) primer and a first-strand cDNA synthesis kit (Pharmacia,
Uppsala, Sweden).
Preparation of genomic DNA from paraffin-embedded biopsy specimens.
Paraffin-embedded tissue was cut using a microtome with a thoroughly
cleaned blade and blockholder. Five to six 5-µm sections from each
sample were transferred to a sterile 1.5-mL Eppendorf vial using a
sterile needle. Paraffin was removed by two separate additions of 0.5 mL of xylene, with incubation at room temperature for 5 minutes,
followed by three washes with stepwise increments of ethanol to a final
wash in 100% ethanol. Protein was digested with proteinase K (400 ng/mL; Sigma, Poole, UK) in a final volume of 400 µL for 6 hours at
45°C. The enzyme was inactivated at 90°C for 5 minutes, and the
vial centrifuged at 15,000 rpm for 20 minutes. The supernatant was used
directly in the subsequent polymerase chain reaction (PCR).
Amplification of VH genes.
For identification of the tumor VH genes, 50-µL PCR
reactions were performed using 2 µL of cDNA or genomic DNA
(gDNA). Mixed 5 -oligonucleotide primers specific for the
VH leader or framework region 1 (FWR1)
sequences27,28 were used together with a consensus 3-JH primer,29 with conditions as
described.28 Seminested FWR2 PCR amplifications were
performed with a consensus 5-FWR2 primer and an outer (30 cycles) and
inner (20 cycles) consensus 3-JH primer.29 At
least two separate PCR amplifications were performed and analyzed for
each sample.
Amplification of constant region genes.
Nested PCRs were performed for identification of constant regions and
tumor-derived isotype variants. The initial amplification from cDNA was
performed with the family-specific VH leader 5-primer, together with an outer 3-constant region primer30,31 (and Table 2). Thirty cycles of amplification
were used, with annealing at 60°C and a final extension at 72°C for
20 minutes. For the second reaction, a patient-specific 5-primer was
designed based on the 3-end of the VH gene segment and the
first half of the CDR3 (Table 2). These primers were used together with
an inner 3-primer30,31 (and Table 2). Primer pairs were
used at 20 pmol/50 µL per reaction, and 20 cycles of amplification
performed under the same conditions as the outer PCR. Each of the five
constant regions was amplified separately, and at least two separate
PCR amplifications were performed for each constant region and biopsy.
Cloning and sequencing of PCR products.
Amplified products were gel-purified, cloned by ligation into pGEM-T
vector, and transfected into JM109 competent bacteria (Promega,
Southampton, UK). Some products from the constant-region second-round
PCRs were ligated directly without purification. Nucleotide sequences
were determined by the dideoxy chain termination method using the
M13-20 and reverse primers. Alignment was made to Entrez and
V-BASE32 databases, using MacVector 4.5.3 software (Kodak
IBI, New Haven, CT).
| |
RESULTS |
Histologic and immunophenotypic analysis of patients' biopsy
specimens.
The clinical and biopsy information is summarized in Table 1. Stained
sections showed extensive infiltration by tumor cells in all cases,
with follicular architecture in the FCL from BA, but sheets of tumor
cells without any follicularity in both cases of DLCL. There was no
significant change in histologic appearance in the different tissues
from each patient during the course of the disease and, specifically,
the presenting biopsy samples of the two DLCL showed no evidence of
transformation from low-grade lymphomas. All tumor cells expressed the
B-cell markers CD20 and CD79a, and were negative for the T-cell markers
CD3 and CD45RO. The proliferative fraction of cells expressing the Ki67
antigen was approximately 20% in the FCL and 60% in the DLCL (Table
3). Expression of Ig by tumor cells could
only be assessed from the frozen sections, and results are shown in
Table 3. All cases expressed monotypic light chains in all tumor cells
in at least one biopsy. However, in the last biopsy of patient EJ
(EJ-3F), light chains were not detected, possibly because of a
technical problem. Monotypic  light chain and dual expression of IgA
(100%) and IgG (>80%) was seen on tumor cells at presentation for
patient BA (BA-1F). However, there were also single IgM+
cells localized within and without the neoplastic follicles, which
could not definitively be assigned to the tumor cell population. For
patient EJ, the presentation biopsy (EJ-1F) showed all tumor cells
expressing IgM, with a proportion also expressing IgD, and some
positive for IgG. This IgG was not detectable in the subsequent two
biopsy samples (Table 3). For patient BR, the tumor cells in the biopsy
specimen at relapse (BR-2F) expressed monotypic  light chains and
both IgM (100%) and IgG (90%) (Table 3). This strongly suggests that
IgM and IgG were expressed on the same cells.
Identification of tumor-derived VH genes.
Predominant repeated VH-DH-JH
sequences with a clonally related CDR3 "clonal signature" were
identified in all cases (Table 4). The
finding of only one group of repeated sequences with identical CDR3
strongly indicates their derivation from the tumor cells.28
Other sequences detected were individually distinct and were likely to
be derived from normal B cells. The same clone was detected at
presentation and in subsequent biopsy specimens for all patients (Table
4). The human VH repertoire is known33-35 and
polymorphic variation in human VH genes generally arises by insertional or deletional changes.34 Therefore, the closest germline sequences for each case could be identified by comparison with
the database. VH genes were derived from VH1
(BA) and VH3 families (EJ and BR) and showed a significant
level of somatic mutation in all cases (Table 4). BR showed the highest
frequency of mutations with 84.6% (BR-1P) and 86.7% (BR-2F) sequence
homology to the closest germline match. Comparison with the closest
germline D-segment genes showed evidence for N-additions in all cases, and JH3a and JH4b genes were used (Fig
1). Nucleotide sequences have been
deposited in the EBML database (accession nos. AJ001404 to AJ001413).
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| Fig 1.
Nucleotide sequence comparison of the tumor-derived
sequences to the closest germline VH, D-segment and
JH-gene shown from CDR2. The numbering indicated above each
case refers to the codons counted from the beginning of VH.
Sequence identity to germline is indicated by dots in each sequence,
each mutation by the appropriate nucleotide. The bottom line in each
instance indicates whether a mutation is silent (lowercase) or has led
to a replacement amino acid (uppercase). N-additions between
VH and D, as well as between D and JH, are
shown in each sequence. Note the stopcodons in position 89 in BA.
|
|
Pattern of somatic mutations and intraclonal sequence variation.
In all cases, somatic mutations were scattered throughout the
VH sequence and, although there were many mutations common
to all clones of each case, there was clear evidence for intraclonal heterogeneity, indicating ongoing mutational activity. The patterns from the three patients are illustrated by the partial sequences (CDR2-CDR3) in Fig 1 (partial sequences are shown to simplify the data
presentation). Each identified clone had a degree of mutational
variation, although in some cases the variant nucleotides cannot be
seen in the figure as they lie outside the CDR2-CDR3 region. The
nucleotide variation observed was greater than expected from the error
rate of Taq DNA-polymerase (~1/5,000 bp in this laboratory). To be
confident of the reality of mutational changes and to remove the
possibility of their being caused by PCR error, only those changes
which occurred in more than one sequence have been considered for
inclusion in Fig 1. However, in both biopsy samples of BA, in all three
from EJ, and in the sample at presentation of BR we found additional
mutations that were not repeated in other sequences. Their frequency
also by far exceeded Taq error rate (BA:  1:500, EJ: 1:900, BR-1P:
1:250) and this greatly increases (over twofold) the number of
unique sequences for these biopsy specimens.
Similar nucleotide variations were seen in the later biopsy samples
from patients BA and EJ (Fig 1), who failed to achieve complete
remissions. For patient BA, an identical stop codon was detected at
residue 88 (GCT  TCT) in a subclone from both the presentation
biopsy sample and that taken at relapse, 10 months later. Because both
RNA and DNA analysis identified only one
VH-DH-JH rearrangement in this
patient and its sequences are therefore likely to be functional, the
nucleotide change in position 88 renders the potentially functional
VH gene nonfunctional. Detection at two timepoints could be
due either to a repeated mutation in this codon, or might indicate that
neoplastic B cells with nonfunctional VH sequences can
survive. In contrast, the second biopsy specimen from patient BR, who
had achieved complete clinical remission after chemotherapy, contained
only a single sequence in 10 of 10 clones, showing a homogeneous
pattern of mutation with no apparent intraclonal variation (Fig 1). The
homogeneity in the BR-2F sequence underlines the reality of the
intraclonal variation observed in the initial biopsy sample from BR and
in the other patients.
To confirm the finding of intraclonal heterogeneity in DLCL, seven
further cases of B-cell DLCL have been studied. As for cases BR and EJ
the samples were presentation biopsies of primary nodal lymphomas
without clinical or histological evidence of transformation from
low-grade lymphoma. Intraclonal heterogeneity was evident in 6 of 7 lymhomas; however, one case was homogeneous with 10 of 10 identical
sequences (unpublished data, December 1997).
The predominant VH sequences of each patient were analyzed
for statistically significant clustering of replacement mutations which
would be indicative of a role for antigen selection.36 No
significant clustering was observed in the CDRs. Evidence for conservation of sequence could be shown in some samples in the FW
regions. In BA the statistical analysis yielded a P value of borderline significance at diagnosis (BA-1F) and in BR P values for sequence conservation reached statistical significance in both
samples (Table 5).
Transcripts of alternative isotypes.
Analysis of transcripts of tumor-derived
VH-DH-JH clonal sequences linked to
Cµ, C , C , C , or C was restricted to frozen material from
which RNA could be isolated. For this, patient-specific 5-primers
complementary to sequences spanning the 3 end of VH to the
beginning of CDR3 were used together with 3-constant region primers
for the selected isotype. In all cases, a product was obtained from the
major isotype expected by immunophenotypic analysis (IgA for BA, IgM
for BR and EJ) (Fig 2). However, products
from additional isotypes were identified for all patients (Table
6). These contained the same CDR3 as
observed in the full sequences obtained from VH to
JH (Figs 1 and 2).
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| Fig 2.
Identification of clonally related multiple isotype
transcripts in the three tumors. Aligned are the tumor-derived
sequences from the patient-specific primer sequence through CDR3 to the constant regions. Sequence identity to germline is indicated by dots in
each sequence, each mutation by the appropriate nucleotide. The
underlined sequences show the patient specific 5 primers constructed
for each case separately.
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For patient BA (Fig 2), there was a mutation in JH (codon
position 102, GAC GAT) that was present in only some of the
clones in Fig 1 and is, therefore, part of the intraclonal variation. Although it occurred in only one sequence from IgA, it was also found
in the IgM and IgG transcripts. In the IgM-derived sequences (Fig 2)
there was identity with those from IgA across the CDR3. Also, the same
mutation in JH (codon position 111, ACC GCC), as well
as intraclonal variation at codon position 102, were observed, confirming derivation from the same B-cell clone. In the constant region of the IgM, there was a deletion of nine nucleotides at the J-C
junction observed in all of the IgM-derived clones, obtained from three
separate PCRs. Other B cells from the same patient did not show this
deletion, suggesting that it reflects a tumor-specific lesion, possibly
in RNA processing. Isotype transcripts of VH-C (IgG2
subclass) were also detected in two of the clones sequenced from a
separate PCR, while the other clones were derived from B cells with
unrelated CDR3s. This indicates the presence of other IgG+
B cells. In the CDR3 a nucleotide change in codon position 102, as
well as changes in codon 108 (GGC GAC) and 111, documented intraclonal variation. No transcripts could be identified from IgD or
IgE in BA.
All three biopsy samples from patient EJ had tumor-derived transcripts
from IgM. These were readily matched to the original VH
sequence due to the long CDR3 sequence, and to two mutations in
JH (Fig 2, codon positions 120 and 121). In addition, the
presentation biopsy specimen yielded transcripts from IgD, IgG
(subclass IgG3) and IgA (Fig 2), all of which had these features.
Transcripts from IgE were also detected in the second biopsy sample
(EJ-2F). The third biopsy specimen (EJ-3F) taken at relapse yielded
abundant VH-Cµ sequences, but no alternative transcripts
were detected (Table 6).
For patient BR, transcripts of VH-Cµ were obtained as
expected from the phenotype. The only alternative isotype detectable was VH-C (IgG3 subclass), found in 5 of 8 clones from
the PCR. Detection of IgG-derived sequence is consistent with IgG
protein expression, and no IgD, IgA, or IgE transcripts were found.
Identity of the IgG3 sequences with the IgM-derived clones was clear
from the CDR3 sequence, and confirmed by finding the same five
mutations in the JH sequence of both isotypes (Fig 2). Two
of these mutations were also documented in all sequences from leader to
JH (codon positions 107, GAC GAT and 108, TAC TAT).
In an attempt to assess the generality of the presence of
alternative isotype transcripts, the presentation biopsy specimen of a further typical primary nodal DLCL, with a reported phenotype IgM, was investigated. Transcripts with a clonally
related VH-Cµ, VH-C, and
VH-C were identified (unpublished data, December
1997), indicating that our three patients were not
exceptional.
| |
DISCUSSION |
Analysis of VH gene sequences is being used increasingly to
provide information about the clonal history of the cell of origin of
B-cell lymphomas. Patterns of somatic mutations within the tumor cell
population can also reveal whether or not the tumor cells are
continuing to mutate after neoplastic
transformation.1,5,20,21 The consequent intraclonal
heterogeneity is commonly found in FCL and might fit with the location
of these tumors in the germinal center where the mutation mechanism is
likely to be activated.4,37 A similar pattern has been
reported in some,7,38 but not all,39,40 cases
of endemic and sporadic Burkitt's lymphoma. However, the majority of
B-cell tumors, including CLL,3 lymphoplasmacytoid tumors,8 and multiple myeloma10,30,41 show
stable homogeneous VH sequences.
Diffuse large cell lymphoma can develop as a primary tumor or after
blastic transformation of FCL.42 In the latter cases, it
has been found that the intraclonal heterogeneous VH
sequences in the FCL can be narrowed to a single sequence, suggesting
that transformation occurred in a single cell.5 Fewer
studies have been made of primary DLCL but, in one study of 17 cases,
no intraclonal heterogeneity was observed, leading to the conclusion
that there was a fundamental difference between FCL and
DLCL.12 Although little clinical information was given, it
was noted that many of the cases were obtained after patients had
relapsed from treatment.12 Therefore, it is possible that
those cases were similar to the second biopsy specimen of our case BR,
who had obtained a clinical complete remission after treatment but who
later relapsed. We found intraclonal heterogeneity only in the
presenting biopsy sample, and a single sequence in all clones at
relapse. The presence of intraclonal heterogeneity in both DLCL at
diagnosis suggests that there is no real difference in mutational
pattern between FCL and DLCL. Furthermore, we have been able to detect
intraclonal heterogeneity in a consecutive series of a further 6 of 7 cases of primary nodal DLCL, confirming that this is a common feature in DLCL (unpublished data, December 1997). Because it is
ethically inappropriate to rebiopsy untreated patients, it is difficult to assess whether duration of disease has an influence on intraclonal variation. However, we can compare successfully treated patients with
those who fail to achieve remission. The latter appear to retain
intraclonal heterogeneity, and in the case of patient EJ, that
heterogeneity was maintained in two biopsy specimens taken at different
timepoints from the same tonsillar site. For patient BR, who relapsed
after complete remission, the primary site had been removed, and the
homogeneous sequence that emerged was from a different tissue site.
Although it is likely that this reflects the selective pressure of
chemotherapy, there remains a possibility that heterogeneity may not be
present at all tumor sites. To avoid possible pertubations introduced
by treatment we would argue that deductions of the clonal history of
the cell of origin of a B-cell tumor should ideally be made from
material at presentation. In our case of FCL and in case EJ,
chemotherapy did not achieve clinical remission, and therefore the
heterogeneous clone continued to exist throughout the course of disease
in both cases.
The availability of the full VH sequences from the tumor
cells allowed us to seek transcripts from constant regions of Ig that
were not obvious from immunophenotypic analysis. In fact the tumors had
been assigned by initial immunophenotyping as IgA (BA) and IgM
(BR and EJ). The additional presence of IgG had been ignored because of
the possibility of contamination from serum IgG in the tissue section,
and this may be a common conclusion. It may have been influenced by
early analyses of CLL, where dual expression of IgM and IgG was often
observed, but was usually due to bound serum IgG.43
However, probing of transcripts is now showing multiple isotypes in the
majority of cases of CLL.15-19 There is a suspicion that
these transcripts are produced by minor cell populations which have
undergone deletional isotype-switching events.19 However,
there is the possibility that alternative transcripts can be generated
by trans-splicing of RNA,44,45 and this is a likely
mechanism in the rare cases of CLL where the cells clearly express both
isotypes.21 Even here, caution in deductions from phenotype
are required, particularly if there is a high level of secreted IgG. In
a recent analysis of IgG-secreting lymphoplasmacytoid tumors, cells
were phenotypically IgM+IgG+, but the clonally
related transcripts from each of the two isotypes had different
patterns of somatic mutations and must, therefore, be from different
cells.46
We randomly chose our three lymphomas based on the availability of
frozen material. With no paraproteins to complicate phenotypic analysis, a more detailed Ig profile was largely consistent with transcript identification, and suggested that for BA
(sIgA+IgG+) and BR
(sIgM+IgG+) a single cell population was
producing at least two isotypes. This points to RNA processing as a
possible mechanism. Even if deletional recombination has occurred on
the productive chromosome, processing could involve constant region
gene transcripts from the allelic chromosome.45 The
situation is less clear for the IgM population in BA which
phenotypically appeared as a minor subset. It is also difficult to
unravel the findings in patient EJ, where transcripts from all isotypes
were evident in the second biopsy specimen, but the cells were
immunophenotypically IgM+ with some (20%) IgD+
only. If subsets synthesizing other isotypes were present, they must
have been so in very small numbers. Interestingly, the alternative transcripts disappeared after combination chemotherapy.
Although the patient samples were taken randomly for investigation, the
common finding of alternative isotype transcripts raises the question
of whether these cases were typical. However, the analysis of a further
cases of DLCL with a reported IgM+ phenotype gave similar
results, indicating that alternative transcripts may occur frequently.
Our data do not show that all transcripts are functional templates for
protein synthesis. Immunohistochemistry is not sufficiently
discriminating to demonstrate expression of alternative Ig isotypes at
low levels or in minor subpopulations, particularly as serum Ig may
interfere. To detect subpopulations of tumor cells that may have
undergone deletional isotype switch, with expression of downstream
isotypes, sorting of cell suspensions will be required. This approach
will also allow separation into single cells, which can then be studied
for potential expression of multiple isotypes.
In summary, immunogenetic analysis of these three cases of lymphoma
taken randomly from our clinical material has revealed two new
findings: the first is that VH gene mutational patterns may
be influenced by disease status. Our limited cases suggest that DLCL at
presentation may still be influenced by the mutation mechanism. The
second finding is that alternative isotype transcripts are not confined
to CLL. Lymphomas of both FCL and DLCL categories also appear to show
signs of isotype-switching events. Whether these represent real
differences in differentiation status or reflect
unexpected products of RNA processing will have to be answered at the
single-cell level.
| |
FOOTNOTES |
Submitted September 9, 1997;
accepted January 20, 1998.
Supported by the Cancer Research Campaign, UK.
Address reprint requests to Christian H. Ottensmeier, MD, Molecular
Immunology Group, Tenovus Laboratory, Southampton University Hospitals,
Tremona Rd, Southampton SO16 6YD, UK.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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I. S. Lossos, A. A. Alizadeh, M. B. Eisen, W. C. Chan, P. O. Brown, D. Botstein, L. M. Staudt, and R. Levy
Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas
PNAS,
August 29, 2000;
97(18):
10209 - 10213.
[Abstract]
[Full Text]
[PDF]
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Abstract
Introduction
Abstract