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V H Gene Analysis of Clonally Related IgM and IgG From Human
Lymphoplasmacytoid B-Cell Tumors With Chronic Lymphocytic
Leukemia Features and High Serum Monoclonal IgG
By Surinder S. Sahota, Richard Garand, Regis Bataille, Alastair J. Smith, and Freda K. Stevenson
An unusual group of human B-cell tumors with cellular
features of chronic lymphocytic leukemia or lymphoplasmacytoid leukemia, together with high levels of a monoclonal
IgG serum protein, has been investigated. Analysis of tumorderived VH genes of neoplastic B lymphocytes was used to
determine the clonal relationship between the IgM expressed or secreted by the tumor cells and the IgG serum
paraprotein. In all five cases, VH gene sequences showed
transcripts of IgM and IgG of common clonal origin. Sequences were derived from VH3 (4 of 5) and VH1 (1 of 5)
families and were all highly somatically mutated with strong
evidence for antigen selection. There was no intraclonal
variation detectable in either IgM or IgG sequences. In 3 of 5
cases, in which monoclonal IgM and IgG were found in
serum, the VH genes combined to Cm or Cg showed identical
mutational patterns. However, in 2 of 5 cases, in which IgM
was confined to cell expression with only monoclonal IgG in
serum, sequences of the VH transcripts of IgM and IgG
showed many shared mutations but also numerous differences. In these cases, the level of mutation was similar in
IgM and IgG and both appeared to be antigen selected. In
summary, the final neoplastic event in this group of tumors
has apparently occurred at the point of isotype switch from
IgM to IgG, leading to dual isotype synthesis. In the group
that secreted both isotypes, the mutation pattern was
identical, indicating either synthesis by a single cell, or
silencing of mutational activity before switching. In the
group that did not secrete IgM, cells of each isotype were
distinct and reflected a divergent mutational history.
r 1998 by The American Society of Hematology.
D
to involve deletional recombination with excision of the upstream constant regions.5 It appears that a considerable degree
of somatic mutation can occur before switching,6,7 leading to
production of IgM memory cells.7 Further mutations may
accumulate after switching,6,7 but the mutational mechanism
appears silent in the fully differentiated plasma cell.
Neoplastic transformation can occur at several points in
B-cell differentiation, from the pre-B cell to the plasma cell.
Investigation of VH genes used by B-cell tumors is now
providing information on the nature of the cell of origin and its
clonal history. For example, it appears that the usage of VH
genes by certain tumors is highly asymmetric, which may be
important for pathogenesis.8-10 Analysis of VH gene sequences
is also indicating whether the cell of origin has entered the
germinal center, known to be the site of somatic mutation.3
In the case of chronic lymphocytic leukemia (CLL), a
proportion of VH sequences are unmutated, consistent with a
naive B cell.11 However, there are subsets of CLL, particularly
those with abnormalities in chromosome 13q14, that have
accumulated significant levels of mutation, indicative of a
different maturation state.12 Follicular lymphoma and Burkitt’s
lymphoma have both undergone somatic mutation as expected,
and mutations continue to accumulate in these tumors subsequent to the final transformation event leading to intraclonal
heterogeneity.13-15 In contrast, splenic lymphoma with villous
lymphocytes16 and myeloma17 have mutated VH sequences, but
no intraclonal heterogeneity, suggesting that the final event in
these tumors is at a postfollicular stage.18
Although it is possible to induce isotype switching in CLL
cells in vitro,19,20 the ability of B-cell tumors to isotype switch in
vivo was difficult to document before the development of VH
gene probes. Coexistence of CLL and an isotype-switched
plasma cell tumor such as myeloma is very rare, and commonly
involves different light chain types, or different idiotypic
antigens, indicative of biclonality.21 In an extensive literature
survey of CLL and the more aggressive tumors that develop in
approximately 10% of patients, genetic relatedness was a
common finding only in cases of prolymphocytic transformation.22 Transformation to myeloma showed clonal relatedness in
only 2 of 8 cases, indicating that this type of disease progression
URING NORMAL B-cell differentiation, a functional
VH-DH-JH transcript is generated by a process of genetic
recombination. A single VH gene is chosen from the available
VH repertoire consisting of approximately 51 potentially functional genes that can be grouped into structurally related
families VH1 to VH7.1 Choice of D-segment gene and imprecision at the joints gives rise to heterogeneity in amino acid
sequence at the third complementarity-determining region
(CDR3). Therefore, the CDR3 sequence can be considered as a
clonal signature for a B cell and, following neoplastic transformation, can act as a useful tumor marker.2
After a similar process of recombination in the light chain, a
naive B cell expresses IgM and can bind antigen. The cell may
then enter the lymphoid follicle and undergo a second process
of sequence diversification by somatic hypermutation.3 Although the introduction of mutations has an element of randomness, the limited antigen in the germinal center will stimulate
only those B cells that offer complementary sequences to
antigen via the contact points in CDR1, CDR2, and CDR3.3,4
Therefore, the imprint of antigen selection will be a clustering
of replaced amino acids in these regions. The next process in a
maturing antibody response is a switch from IgM to another
isotype, often IgG. The switching mechanism has been shown
From the Molecular Immunology Group, Tenovus Laboratory, Southampton University Hospitals, Southampton, UK; and the Centre
Hospitalier Universitaire de Nantes, Institut de Biologie, Nantes Cedex,
France.
Submitted June 5, 1997; accepted August 28, 1997.
Supported by the Leukaemia Research Fund, UK, and the European
Myeloma Research Network (Biomed BMH1-CT93-1407).
Address reprint requests to Freda K. Stevenson, PhD, Molecular
Immunology Group, Tenovus Laboratory, Southampton University
Hospitals, Tremona Road, 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’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9101-0032$3.00/0
238
Blood, Vol 91, No 1 (January 1), 1998: pp 238-243
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VH GENES IN IgG-SECRETING LYMPHOPLASMACYTOID TUMORS
arises relatively rarely via clonal evolution,22 a finding supported by more recent cases.23 However, transcripts of clonally
related VH-Cµ have been found in patients with myeloma,
which point to coexistence of a minor population of less mature
B cells.24,25
We have had the opportunity to study an unusual subset of
B-cell tumors with abnormal cells expressing or secreting both
monoclonal IgM and IgG. Such cases are likely to be closer to
lymphoplasmacytoid lymphoma (LPL) than to true CLL, although some share features with both entities. LPL is a distinct
B-cell proliferative disorder with particular morphological and
histopathological characteristics, and is frequently associated
with a high secretion of monoclonal IgM.26 However, cases of
CLL may also exhibit high levels of monoclonal Ig, usually
IgM, and can show morphological or phenotypic lymphoplasmacytoid differentiation.27 On the other hand, cases of LPL may
have blood and bone marrow involvement with cells difficult to
distinguish from CLL.26 Our cases appear closer to LPL than
CLL and have the additional rare feature of synthesizing both
IgM and IgG. Because they may, therefore, reflect the point of
isotype switch, we have used immunogenetic analysis to show
the clonal history of these tumors.
MATERIALS AND METHODS
Patient selection. The focus of this investigation was on patients
with B-cell tumors who had evidence for synthesis of both IgM and
IgG. From January 1989 to May 1996, we investigated in the
Department of Hematology in Nantes, France, 611 cases of B-cell
lymphoproliferative disorders (BLPD), with circulating abnormal lymphoid cells .5 3 109/L, and CD19 .30%. Among these, we observed
12 patients with a high serum monoclonal IgG. Four of these patients
had the same light chain type in IgM and IgG and were selected for
further study. An additional patient (BAR) presented in Southampton
with renal stones, and was found to have IgM and IgG paraproteins of
similar light chain type in serum. He had a normal blood count and has
remained well for 6 years since diagnosis.
Morphological and histological classification of BLPD. All MayGrünwald-Giemsa stained blood and bone marrow smears were reviewed and classified according to the French-American-British (FAB)
group proposals.28 Similarly, all biopsy specimens were reviewed and
classified according to the revised European-American classification of
lymphoid neoplasms (REAL).26
Immunophenotypic analysis. Peripheral blood mononuclear cells
were prepared by separation on a Ficoll gradient and analyzed by
indirect immunofluorescence, using a panel of monoclonal antibodies
(MoAbs), including B3 and B6 (CD22 and CD23, respectively; Coulter,
Miami, FL), leuM5 (CD11c) and B-B4 (CD138), a SYNDECAN-1
plasmacyte-specific MoAb29 (Diaclone Research, Besancon, France),
using the FACSCAN (Becton Dickinson, San Jose, CA). Coexpression
of CD5 and CD19 was assessed by double labeling using fluorescein
isothiocyanate (FITC)-labeled IOT1a (CD5) and phycoerythrin (PE)labeled IOB4 (CD19; Immunotech, Marseille, France). Surface Ig (sIg)
expression was studied with FITC-labeled F(ab8)2 from goat polyclonal
antibodies against human Igµ, Igd, Igg, Igk, and Igl chains. sIg
fluorescence intensity was considered as strong when the difference
between mean fluorescence of both anti-light chain types exceeded one
log.
Analysis of VH genes. Total RNA (5 to 50 µg) was isolated using
RNAzol (Cinna Biotecx Labs Inc, Houston, TX) from mononuclear cell
fractions prepared either from heparinized peripheral blood and stored
as cryopreserved cells in dimethyl sulfoxide (0.9 to 1.5 3 107 cells;
patients PAI, BLO, SAM, and LAR), or from a heparinized bone
239
marrow aspirate (patient BAR). RNA (2 to 10 µg) was reverse
transcribed to cDNA with Cµ and Cg isotype-specific primers as
described.30 A sample of cDNA was then amplified by the polymerase
chain reaction (PCR) using a mixture of 58-oligonucleotide primers that
cover the VH1 to VH7 family potentially functional repertoire, together
with nested downstream 38-primers specific for the CH isotype. Amplification conditions and methods of cloning and sequencing of products
have been described.30
RESULTS
Clinical and laboratory features of patients. All patients
had evidence of B-cell tumors that were synthesizing IgM and
IgG, but the detailed nature of the tumors was variable. Four of
five patients presented with a significant lymphocytosis with
cell morphology typical of CLL. However, a few coexisting
lymphoplasmacytoid cells were observed in two patients (BLO
and SAM). One of five patients (BAR) had no lymphocytosis
but had tumor cells in the bone marrow with morphology
consistent with LPL. Histologic data were available in three of
five cases (Table 1). According to the REAL classification, two
patients showed a histopathologic picture of LPL, one in a
bronchial biopsy specimen (BLO), the other in bone marrow
and in spleen (red pulp infiltrate; SAM). A third patient (LAR)
had an amyloid immunoglobulin light chain (AL)-type amyloid
nephropathy on a kidney biopsy specimen. All patients had high
levels of monoclonal IgG paraproteins in serum, and in one case
(PAI) two IgG paraproteins were detected. Patients PAI, BLO,
and BAR also had an additional IgM paraprotein of the same
light chain type as the IgG (Table 2). In four of five cases,
circulating tumor cells were present, and phenotypic analysis
(Table 3) showed increased numbers of CD191 lymphocytes.
Only one case (PAI) showed an immunophenotypical profile
characteristic of CLL (CD51 CD221 CD231), and these cells
had an unusual strong sIg (11) expression (Table 3). Patients
BLO, SAM, and LAR exhibited a CD52 CD221 CD231/2,
sIg11 surface phenotype more typical of LPL.26,31 The four
cases with tumor cells in the blood were all negative for CD10,
CD11c, and B-B4.
Circulating lymphoid cells expressed both sIgM and sIgG
with similar light chain type in three patients (PAI, SAM, and
LAR), but only IgM in patient BLO. In the case of BAR, only a
bone marrow aspirate was available, and this had the cytological features of LPL. The tumor cells expressed CD19, and
Table 1. Clinical and Laboratory Features of B-Cell Tumors With High
Serum IgG Paraprotein Levels
Circulating
Classification
Age
Binet Lymphocytes
29
Patient (yr) Splenomegaly Stage
(310 /L)
Morphology Histology
PAI
BLO
BAR
SAM
LAR
68
83
69
44
70
1
1
2
1
2
A
B
ND
A
A
15.7
8.9
Normal
5.6
9.1
CLL
CLL
LPL†
LPL
CLL
ND
LPL*
ND
LPL‡
AL A§
Abbreviations: CLL, chronic lymphocytic leukemia; ND, not determined; LPL, lymphoplasmacytoid lymphoma; AL A, AL amyloidosis.
*Bronchial biopsy.
†Bone marrow.
‡Bone marrow and lymph node.
§Amyloidosis on kidney biopsy.
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240
SAHOTA ET AL
Table 2. Serum Paraprotein Analysis
IgG
IgM
Patient
Type
Concentration (g/L)
Type
Concentration (g/L)
PAI
BLO
BAR
SAM
LAR
2 3 IgGk
IgGl
IgGl
IgGk
IgGk
20
20
10
40
21
IgMk
IgMl
IgMl
—
—
5
6
3.6
—
—
analysis of cytoplasmic Ig showed an IgMGk1 population.
However, in all cases, conclusions concerning dual expression
of IgM and IgG were limited by the presence of high serum IgG
paraprotein, which may be bound to Fc receptors.32
VH genes of tumor-derived VH-Cµ and VH-Cg transcripts.
Identification of expanded tumor-derived VH sequences from
repeated identical or closely similar CDR3 ‘‘clonal signature’’
sequences, obtained by PCR/cloning, has been validated previously.33 In all cases, amplification of VH in combination with Cg
yielded repeated sequences with clear CDR3 ‘‘clonal signatures’’ (Table 4). Tumor-derived deduced amino acid sequences
are shown in Fig 1. Nucleotide sequences have been deposited
in the European Molecular Biology Laboratory (EMBL) database (accession numbers: Z95481-95487). Other VH sequences
obtained were individually different and were likely to be from
normal B cells. For all patients, the same VH sequence was also
found to predominate in sequences combined to Cµ (Fig 1),
indicating derivation of both IgG and IgM from the same
original B cell. Most of the tumors used VH genes from the VH3
family (4 of 5), with one out of five from VH1. The CDR3
sequences were short (6-14 amino acids), particularly when
compared with typical CLL,11 and a range of JH genes was used,
some of which were somatically mutated (Fig 1). There was no
evidence for intraclonal variation in any of the tumor-derived
sequences. All the IgG products were derived from the IgG1 or
IgG2 subclass (data not shown); further assignment could not
be made from the limited available length of constant region
nucleotides.
In the case of PAI, although two IgG paraproteins were
observed in serum, only a single VH-Cg transcript was identified, suggesting that either the two IgG serum paraproteins were
clonally identical, or that the second paraprotein was being
produced from a separate cell population. In contrast, two
different repeated VH-Cµ transcripts were found for PAI, one of
which was identical to the IgG-derived sequence (Table 4). The
VH of the second IgM-derived transcript used the V4-59 gene
segment but contained a stop codon at position 47 rendering it
Table 3. Immunophenotype of Tumor Cells
sIg
Patient
PAI
BLO
BAR
SAM
LAR
CD19
80
88
16
48
73
%
Type
CD5
CD22
CD23
CD38
78
73
19
48
67
MGk11
86
1
ND
13
20
47
82
ND
48
34
37
46
ND
44
0
ND
0
ND
22
6
Ml11
MGl*
MGk11
MGk11
Tumor cells were obtained from blood, except for patient BAR in
whom a bone marrow aspirate was analyzed.
*Cytoplasmic Ig.
aberrant. This aberrant sequence was likely derived from the
allelic chromosome of the tumor cells, and had apparently not
undergone isotype switching.
Analysis of somatic mutations. To assess the degree of
somatic mutation, the VH sequences were compared with the
closest germ line sequences in the databases. Although this
approach does not allow for sequence polymorphism, it has
become clear recently that the majority of polymorphic changes
in human VH genes arise from deletions or insertions, and that
sequence variation is at a low level.1 The percent homology of
the VH sequences with the closest germ line genes was quite low
(Table 4) indicative of extensive somatic mutation. Deduced
amino acid sequences (Fig 1) show the positions of the
mutations and indicate amino acid replacements. Comparison
of the pattern of mutations in the sequences derived from IgG
with those from IgM showed complete identity for patients PAI,
BLO, and BAR (Fig 1A). In contrast, the pattern of mutation in
IgG and IgM from patients SAM and LAR showed some
common mutations but many differences, particularly in the
CDRs (Fig 1B). However, the level of mutational activity was
similar for both IgG and IgM, and there was no evidence for
accumulation of mutations in switching from IgM to IgG. In
fact, at several codons, there were mutations in the IgM-derived
sequences, which were not found in the IgG sequences.
There was an apparent clustering of replaced amino acids in
CDR1 and CDR2 in all sequences, and this was assessed for
statistical significance, which points to antigen selection, using
the method of Chang and Casali.34 The results (Table 5) show
highly significant clustering of replacement amino acids in the
CDRs for IgG- and IgM-derived sequences from all patients.
Where mutations differed between IgG and IgM, both had
strong evidence for antigen selection. There was also evidence
for preservation of sequence in all the functional framework
regions (FWRs; Table 5). A different pattern was observed in the
nonfunctional IgM-derived V4-59 sequence from patient PAI
(Table 5). In this aberrant gene, there was no clustering of
somatic mutations in CDRs indicative of antigen selection, nor
was there evidence for conservation of sequence in the FWR.
DISCUSSION
Assessment of the maturational status of B-cell tumors has,
until recently, been based largely on morphological and phenotypical features. LPL is considered to arise from a mature B cell,
often with cytoplasmic Ig, and commonly associated with a
monoclonal serum paraprotein, usually of IgM type.26 Waldenstrom’s macroglobulinemia is the most common lymphoma in
this category and has been described as a distinct disorder of
small lymphocytes showing maturation to plasma cells.26
However, many B-cell tumors, including CLL, also show some
maturation to plasmacytoid cells. These observations suggest
that B-cell tumors are not completely ‘‘frozen’’ at a point of
differentiation.
Analysis of VH and VL genes is allowing further probing of
the differentiation status of B-cell tumors. Because the V-gene
germ line repertoire is largely mapped,1 it is possible to
investigate the degree and pattern of somatic mutations that
have occurred in the cell of origin. This process is activated in
the germinal center; therefore, accumulation of mutations
indicates that the cell has encountered this site.3 However,
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VH GENES IN IgG-SECRETING LYMPHOPLASMACYTOID TUMORS
Table 4. Analysis of Tumor-Derived VH Genes
Ig
VH
GL
Patient Class Family Donor
PAI
IgG
IgM1
IgM2
VH3
VH3
VH4
BLO
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
VH3
VH3
VH1
VH1
VH3
VH3
VH3
VH3
BAR
SAM
LAR
JH
V3-23 JH5a
V3-23 JH5a
V4-59 (stop
codon)
V3-7 JH5b
V3-7 JH5b
V1-46 JH4b
V1-46 JH4b
V3-74 JH6c
V3-74 JH6c
DP-58 JH3b
DP-58 JH3b
TumorDerived
Clones
%
Homology Clones Sequenced
88.9
88.9
16/22
5/12
5/12
7
5
4
93.2
93.2
95.9
95.9
90.1
90.9
91.2
90.5
7/12
9/12
12/12
10/10
9/9
6/8
6/8
8/8
7
6
12
10
6
4
6
6
Abbreviation: GL, germline.
mutational activity can continue after neoplastic transformation,
as seen clearly in follicular lymphoma in which clonally related
cells have distinct intraclonal sequence variation.13,14 The
distribution of mutations may also indicate a role for antigen
selection, and, because antigen may persist,35 it could stimulate
growth and differentiation of tumor cells.36 The cases we
describe were characterized by circulating lymphoid cells with
CLL morphology, but shared histopathologic and immunophenotypical features of LPL; thus, they may correspond rather to
‘‘lymphoplasmacytoid’’ leukemia than to true CLL. They have
accumulated somatic mutations as expected from a mature cell,
which has evidently passed through the germinal center. In fact,
clustering of replacement amino acids in CDR1 and CDR2 is
even more obvious than in the closely related splenic lymphoma
with villous lymphocytes16 or in follicular lymphoma,13,14
Burkitt’s lymphoma,15 or myeloma.17,18,30,36,37 This significant
clustering provides strong evidence for antigen selection up to
and possibly beyond the final neoplastic event.
Isotype switch events in normal B cells appear to occur at
about the same time as somatic mutation6 leading commonly,
but not invariably, to mutated sequences in IgG1 cells.38 A
similar heterogeneity in mutational frequency is seen in the rare
Fig 1. Deduced amino acid sequences of VH-Cg and VH-Cm transcripts from patients’ tumor cells
synthesizing both IgG and IgM
isotypes. (A) Cases with both
IgM and IgG serum parapoteins.
(B) Cases with IgG serum paraproteins only. Comparisons are
made with the closest germ line
VH genes. Upper case, replacement mutations; lower case, silent mutations. Replacement mutations in JH are underlined.
241
cases of IgG1 or IgA1 CLL.39-41 Interestingly, in some cases of
IgG1 CLL, transcripts of Cµ and Ca combined to the tumor
VH-DH-JH sequence have been observed.42 This suggests either
that minor populations of cells with variant isotype switch
products exist, or that a single population can generate alternative transcripts by trans-splicing of RNA.43 In one report, the
fact that tumor cells expressed dual sIgG and sIgA would
support the latter mechanism.40 However, the argument for
separate populations is supported by detection of a few mutational differences in transcripts in some cases42 and by the
demonstration of deletional bi-allelic recombination in three
cases of isotype-switched CLL.40
A further surprising finding is that blood lymphocytes from
cases of conventional IgM1 CLL contain transcripts of tumor
VDJ combined to a variety of alternative isotypes and that cells
can be induced to synthesize the switched Ig.20,44,45 Thus,
although isotype switching occurs rarely in CLL, transcripts are
present. It is not known at present if these are produced by the
bulk population or from minor populations that have undergone
deletional switch recombination, although again there is some
evidence for the latter.45
The cases we describe appeared to be arrested at a more
mature point in differentiation. They all had high levels of
monoclonal serum IgG, and, in three of five cases, an additional
monoclonal IgM. Analysis of the VH genes of these three cases
showed transcripts of identical VDJ sequences combined to
both Cµ and Cg. For PAI and BAR, there was no phenotypical
evidence for two populations, but this cannot be ruled out. For
BLO, only IgM expression was found, suggestive of a separate
undetected minor population of isotype-switched cells. The
only unequivocal way of confirming isotype heterogeneity
among cell populations with identical VH sequences would be to
separate out individual tumor cells and perform PCR at the
single-cell level, and this is in progress. In the other two cases,
transcripts of VDJ-Cµ and VDJ-Cg were also obtained with
CDR3 similarity indicating clear common clonal origin, but
there were numerous distinct mutations. For these cases, the
IgG1 and IgM1 populations must be separate. This is in spite of
the finding that the cells appeared to coexpress IgG and IgM,
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242
SAHOTA ET AL
Table 5. Distribution of Mutations in the Tumor-Derived VH
Sequences
Patient
Ig Clone
GL Donor
PAI
IgG/IgM1
V3-23
V4-59
BLO
IgM2
(aberrant)
IgG/IgM
BAR
IgG/IgM
V1-46
SAM
IgG
V3-74
IgM
V3-74
IgG
DP-58
IgM
DP-58
LAR
V3-7
R:Sobs (CDR)*
R:Sobs (FWR)
2.8 (14:5)
1.0 (7:7)
4.5 (9:2)
10.0 (20:2)
5.0 (10:2)
1.0 (4:4)
` (4:0)
3.0 (6:2)
4.3 (13:3)
1.6 (8:5)
2.8 (11:4)
1.0 (6:6)
12.0 (12:1)
1.6 (8:5)
16.0 (16:1)
0.6 (4:7)
Rexp (CDR)
6.0
6.0
4.0
2.0
5.0
5.0
5.0
5.0
B cells and has shown that the outcome can be two clonally
related tumor populations.
P (CDR)†
P (FWR)
,.001
,.001
.100
.120
,.001
,.001
.001
.026
,.001
,.001
.002
,.001
,.001
,.001
,.001
,.001
Abbreviations: GL, germline; CDR, complementary-determining region; FWR, framework region.
*R:S is the ratio of replacement (R) to silent (S) mutations observed
in CDRs or FWRs, as compared with those expected by chance alone.
†Probability of obtaining the number of R mutations by chance
alone. P , .05 is considered significant for either excess of R
mutations (CDR) or lack of R mutations (FWR).
and confirms the dangers of phenotypic analysis of sIg32
particularly in the presence of high levels of serum IgG. All
sequences had strong evidence for clustered replacement mutations characteristic of antigen selection, a feature lacking in the
aberrant allelic VH gene identified in one of the patients.
Therefore, it appears that the transformation event occurred at
the level of an IgM1, somatically mutated, antigen-selected B
cell, with characteristics of a memory B cell. The transformed
cell proliferated giving rise to a neoplastic clone, which, in two
cases, secreted IgM. This cell might be analogous to tumor cells
of Waldenstrom’s macroglobulinemia in which Vk-genes have
been found to be somatically mutated.46 However, in our cases,
the cell was also able to undergo isotype switching to IgG, and a
second tumor population was able to proliferate and differentiate to high level IgG secretion. In the IgM-secreting tumors, if
the IgM and IgG are produced from different cells, the mutation
mechanism must have been silenced before switching. In
tumors not secreting IgM, mutation continued, possibly in both
isotypes. This continuing mutational activity led to mutational
divergence, with accumulation of some mutations in the IgMderived sequences that were not found in the IgG-derived
sequences. Curiously, no intraclonal variation was detectable,
indicating that the neoplastic cells represent progeny of only
single IgG and IgM variants. It also suggests that the current
IgM1 population has lost the ability to switch. These tumors
appear to have undergone neoplastic transformation at the
pivotal point of isotype switch from IgM to IgG. They may in
fact represent the bifurcation leading to memory cells or plasma
cells,47 and the IgM1 cells could relate to the precursor cell
identified in some cases of myeloma.24,25 Therefore, immunogenetics has supported morphology in showing that B-cell tumors
can respond to differentiation signals in the same way as normal
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1998 91: 238-243
VH Gene Analysis of Clonally Related IgM and IgG From Human
Lymphoplasmacytoid B-Cell Tumors With Chronic Lymphocytic Leukemia
Features and High Serum Monoclonal IgG
Surinder S. Sahota, Richard Garand, Regis Bataille, Alastair J. Smith and Freda K. Stevenson
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