Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1070-1076
VH Gene Analysis of IgM-Secreting Myeloma
Indicates an Origin From a Memory Cell Undergoing Isotype Switch
Events
By
Surinder S. Sahota,
Richard Garand,
Razeen Mahroof,
Alastair Smith,
Nadine Juge-Morineau,
Freda K. Stevenson, and
Regis Bataille
From the Molecular Immunology Group, Tenovus Laboratory, and
Department of Haematology, Southampton University Hospitals,
Southampton, UK; and the Laboratoire d'Hematologie, Institut de
Biologie, Centre Hospitalier Universitaire de Nantes, Nantes, France.
 |
ABSTRACT |
IgM-secreting plasma cell tumors are rare variants of typical
isotype-switched multiple myeloma with a similar disease outcome. To
probe the origin and clonal history of these tumors, we have analyzed
VH gene sequences in 6 cases. Potentially functional tumor-derived VH genes were all derived from
VH3, with the V3-7 gene segment being used by 4 of 6. All were somatically mutated, with a mean deviation from germline
sequence of 5.2% (range, 3.1% to 7.1%). The distribution of
replacement mutations was consistent with antigen selection in 4 of 6 cases, and no intraclonal heterogeneity was observed. Clonally related
switched isotype transcripts were sought in 4 cases, and C
transcripts with tumor-derived CDR3 sequence were identified in 2 of 4. These findings indicate that IgM-secreting myelomas are arrested at a
postfollicular stage at which somatic mutation has been silenced.
Isotype switch variants show the cell of origin to be at the IgM to IgG
switch point. These features indicate that the final neoplastic event
has occurred at a stage immediately before that of typical
isotype-switched myeloma. One possibility is that IgM myeloma involves
the previously identified precursor cell of typical myeloma.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
NORMAL B CELLS UNDERGO a series of
recombinatorial and mutational changes during differentiation that lead
to unique sequences in the variable region genes, VH and
VL.1 These sequences provide clonal markers for
tracking B-cell clones, and they also reflect the point reached in the
differentiation process.2 V-gene sequences preserved in
neoplastic cells therefore indicate features of the cell of origin and
its clonal history.3 For certain B-cell tumors, such as
those involved in cold agglutinin disease,4 there is
evidence for bias in usage of VH genes that might reflect
stimulation of the cell of origin by a B-cell
superantigen.5 Analysis of somatic mutational patterns has
also shown if the cell of origin has been exposed to the somatic
mutational mechanism, which is generally activated in the
germinal center.6 For example, in chronic lymphocytic
leukemia (CLL), evidence from VH genes has shown
heterogeneity in the cell of origin, with 1 subset derived from a naive
cell with unmutated sequences and another from cells that have
undergone somatic mutation.7 Cases of follicular lymphoma
and other tumors located in the germinal center also have somatically
mutated V-genes.8-10 Interestingly, these tumors often
display intraclonal heterogeneity resulting from ongoing mutational
activity posttransformation.8,10
Normal somatically mutated IgM+ B cells reach a crossroad
in the germinal center from which they can either escape into the blood
as memory cells11 or undergo isotype switch and
differentiation to plasma cells.12 The pathways followed
are controlled by a regulatory network of largely T-cell-mediated
influences, including CD40 ligand and cytokines.13,14 In
neoplastic cells, transcripts of isotype switch variants have been
observed in typical IgM+ CLL,15,16 and there is
evidence for alternative transcripts in cases of follicular lymphoma
and diffuse large-cell lymphoma.17 These findings suggest
that tumor cells are capable of responding to differentiation signals
in vivo.
Analysis of more than 100 VH genes in typical
isotype-switched multiple myeloma (MM) has shown only minor bias in
usage when compared with the normal expressed
repertoire.18-20 A notable feature is that the
V4-34 gene, which is known to be rearranged in 5% to 10%
of normal B cells, has been found in only 1 case of MM, and that case
had an unusual IgD paraprotein.21,22 Myeloma V-genes are invariably mutated and display clustering of replacement amino acids characteristic of antigen selection in approximately 24%
of VH gene sequences.20 This reaches
approximately 70% when analysis includes VL
sequences.23 Lack of intraclonal heterogeneity is another
common feature, and stability of tumor sequence is observed from
presentation to plateau phase of disease.24 These features
indicate that the final malignant event has occurred in a
postfollicular cell. One intriguing question that has remained concerns
the existence of a less differentiated precursor cell of the myeloma
clone, because VDJ-Cµ transcripts have been identified with sequence
identity to the tumor IgG or IgA clone.25-27 However, both
the neoplastic potential of the cells producing these transcripts and
their existence as separate cells remain in question. Difficulties in
identifying the Cµ transcripts would also suggest that these cells
are present at low frequency.27,28
In this study, we have analyzed VH genes of a rare subset
of MM that secretes IgM. The question posed was whether these tumors arise from plasma cells generated before somatic mutation or whether they have accumulated mutations in a manner similar to the previously identified precursor cell of isotype-switched MM. Their maturation status was further assessed by analysis of variant transcript synthesis.
| |
MATERIALS AND METHODS |
Patient material.
Bone marrow aspirates were obtained from 5 IgM MM and 1 IgM
primary plasma-cell leukemia at the time of diagnosis. The
presenting features of these patients are outlined in
Table 1. The distinctive cytological
features of these patients are shown in
Fig 1. In Table 2, these features are contrasted with
those of a cohort of 31 patients with IgM-secreting Waldenstrom's
macroglobulinemia (WM).
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| Fig 1.
Morphology of myeloma cells on
May-Grünwald-Giemsa-stained bone marrow smears (original
magnification × 1,000). Each patient is designated by initials, and 2 micrographs per patient are shown (numbered 1 and 2).
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Cell preparation and phenotypic analysis.
Heparinized bone marrow aspirates were taken and used for
May-Grünwald-Giemsa-stained smears. Bone marrow mononuclear
cells (MNCs) were separated by centrifugation on Ficoll-Hypaque and immunophenotyped as previously described.30
Genotypic analyses.
Cytogenetic analyses and probes and protocols used for fluorescent in
situ hybridization (FISH) and Southern blot analyses were as
described.30a
Preparation of cDNA.
RNA was used as source material from tumor cells to amplify
VH genes. This is a preferred approach to identify
functional transcripts, because it reduces the likelihood of amplifying
the aberrantly rearranged allele. Total RNA (5 to 10 µg) was isolated from the MNC fraction (4 to 10 × 106 cells) of the
bone marrow aspirate using RNAzol B (Cinna Biotecx Labs, Inc, Houston,
TX). Reverse transcription was performed using approximately 2 µg RNA
and an outer Cµ1 constant region primer (Table 3) with a first-strand cDNA
synthesis kit (Pharmacia, Uppsala, Sweden) according to the
manufacturer's instructions. For IgG transcripts, a C1 primer was
used to prime cDNA synthesis, and for IgA transcripts, a C
1 primer
was used (Table 3).
Amplification and sequencing of VH genes.
For analysis of the VH of tumor cells, one fifth to one
third of a sample of cDNA was amplified by polymerase chain reaction (PCR) using a mixture of 5'-oligonucleotide VH leader
primers specific for VH families 1 through 7 and a
downstream nested Cµ2 primer (Table 3). For each sample, PCR
conditions, cloning of amplified DNA, and sequence analysis were as
previously reported.31 Analysis of V-gene sequences was by
alignment to current EMBL/GenBank and V-BASE32 sequence
directories using MacVector 4.0 software (International Biotechnologies
Inc, New Haven, CT). At least 2 independent PCR amplifications were
performed from each sample.
Investigation of tumor-related VH-C/-C
transcripts.
A 2-step nested PCR approach was used to investigate tumor-derived
CH variant transcripts for patients CLE, DEZ, JON, and MOR.
In step 1, one fifth of the cDNA was amplified using the appropriate
5'-VH leader primer together with C1 or C1. In
step 2, 1/20 of the PCR product of step 1 was directly amplified using a tumor-specific CDR2 (for CLE) or CDR3 (for DEZ, JON, and MOR) 5'-primer together with a nested downstream C2 primer or a
C2 primer (Table 3). Amplification conditions were modified to
include an annealing temperature of 65°C for 1 minute in step 2. PCR products of predicted size were cloned and sequenced.
| |
RESULTS |
Tumor cell analyses.
The clinical and phenotypic data of the 6 IgM MM patients analyzed are
summarized in Tables 1 and 2. It is noteworthy that 3 of 6 patients
presented lytic bone lesions on bone radiography, with 1 of these
patients also having hypercalcemia (Table 1). Cellular morphology
assessed by stained bone marrow smears showed typical infiltrating
plasma cells (Fig 1). Clearly, the 6 IgM MM patients had an excess of
malignant plasma cells within the bone marrow, with a median value of
16% (range, 12% to 50%) versus 2% (range, 0% to 10%) in
Waldenstrom's disease (P < .01; Table 2). No expansion of
lymphoid or lymphoplasmacytoid cells was observed, as compared with WM
(Table 2). Immunophenotypic analysis showed the IgM MM tumor cells to
be surface CD38++BB4+ and
CD19
, confirming involvement of mature plasma cells
and excluding any misdiagnosis with B-cell lymphoma (data not shown).
Availability of material allowed cytogenetic analyses in 2 patients
(BEC and MOR). In both patients, a t(11,14q32) translocation event was identified, which was further confirmed by FISH analysis in patient MOR. Southern blot analysis established the translocation breakpoint in
patient MOR as occurring in the JH region and not in the
switch region (data not shown).
VH gene use by tumor cells of patients.
The identification of tumor-derived VH genes was based on a
common CDR3 signature sequence among multiple clones sequenced from
each patient's amplified cDNA.33
Table 4 shows the number of tumor-derived
clones identified of the total number of clones sequenced. Sequences
not related to the tumor clone differed individually and were most
likely derived from normal B cells. The VH gene used by
each tumor, together with deviation in homology from their germline
counterparts, are also shown in Table 4. The deduced amino acid
sequences are shown in Fig 2. Nucleotide sequences have
been deposited in the EMBL database (accession numbers
AJ238036-AJ238040).
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| Fig 2.
Deduced amino acid sequences of VH-Cµ
transcripts from patients' tumor cells. Comparisons are made with the
closest germline VH genes. Uppercase letters, replacement
mutations; lowercase letters, silent mutations. Replacement mutations
in JH are underlined.
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VH gene segments of 6 of 6 were derived from the
VH3 family, and the individual germline genes used were
V3-7 in 4 of 6 and V3-73 and V3-74 each in 1 case. The CDR3 length
(6 to 10 amino acids) in these IgM tumors appears to be at
the shorter end of reported length of CDR3s (2 to 25 amino acids) in
functionally rearranged VH sequences from normal peripheral
B cells.34 The CDR3 of patient CLE apparently lacked a
D-segment gene and consisted of 7 amino acids that were part of
JH6b, which is a long JH gene segment (Fig 2).
Rare functional VH sequences from normal B cells in which
CDR3 is derived from the JH gene segment have been
previously reported.34 D segment genes of origin were not
identified in any of the sequences using the recently established
criteria of Corbett et al.35 Four of 6 cases used the
common JH4b gene.34
Analysis of somatic mutations.
A significant degree of somatic mutation was evident in each tumor
sequence (Table 4 and Fig 2). An average of 5.2% (range, 3.1% to 7.1%) deviation from germline was observed. Assessment of an
influence of antigen selection on VH gene sequence has
previously been based on a binomial distribution analysis of
replacement and silent mutations.36 However, recent data
from patterns in normal B cells have shown significant clustering of
replacement mutations in CDRs even in nonfunctional
sequences.37 Some of this reflects accumulation of
mutations at hotspots.38 The main feature that
distinguishes functional from nonfunctional sequences appears to be
conservation of FR sequences.39 In the IgM myeloma sequences, hot spot mutations were observed in CDR1 (Ser 31) and in
CDR2 (residue 50), as shown in Fig 2. Frequent mutations were also
observed in FR3 at residues 79 and 93. Conservation of FR structural
integrity was assessed using overall R:S ratios, and in 4 of 6 cases
this ratio in FRs was less than 1.7 (Table 4), which is indicative of
negative selection of R mutations.39
An assessment of intraclonal homogeneity among the tumor-derived
clones, identified by shared CDR3 sequence, was made from at least 6 clones, except in the case of the previously published BAR
VH sequence, in which only 3 clones were fully
sequenced.29 A uniform feature of all the IgM tumors
examined was a lack of intraclonal variation, as evident from identical
VH gene sequences from tumor-derived clones.
Investigation of tumor-related isotype switch variants.
In 2 of 4 available cases, tumor-derived CDR3-C transcripts were
identified (DEZ and MOR), and in both cases, VDJ joined to C1 and to
C2 were observed (Fig 3). Sequences from
multiple clones of each variant transcript displayed intraclonal
homogeneity and identity to CDR3-Cµ sequences. In all 4 cases, C
transcripts linked to tumor VDJ were not found using a method
previously shown to identify tumor-derived variant C transcripts in
lymphoma cDNA.17
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| Fig 3.
Nucleotide and deduced amino acid sequences of the
CDR3-C transcripts from patients DEZ and MOR. The number of clones
showing identical sequence are indicated in parentheses and comparison
has been made with the tumor CDR3-Cµ transcript. The position of the
5'-CDR3 primer is indicated, and the position of the downstream
C primer (not shown) allowed identification of C1 or C2
transcripts.
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| |
DISCUSSION |
In this investigation of IgM-secreting myeloma, VH gene
analysis has been used to probe the maturation status and stage of arrest of the neoplastic cell of origin. A high level of somatic mutation was a consistent feature of these IgM-secreting tumors, indicating that the cell of origin has traversed the germinal center
and activated the somatic hypermutation mechanism. Although the number
of cases in this study is small, the level of mutation appears lower
(average 5.2% deviation from germline; range, 3.1% to 7.1%) than in
114 cases of typical Ig class-switched myeloma (8.9%; range, 2.5% to
22.4%).19-23 The level in the IgM-secreting tumors
appeared closer to mutational frequencies in preswitched IgM memory
cells11 and in IgM tonsillar cells.40 The
pattern of somatic mutations in tumor VH genes was
indicative of replacement events in CDR1 and CDR2; however, intrinsic
mutational hot spots may mask analysis of antigen-specific positive
selection.37-39 Nevertheless, significantly low overall R:S
ratios in FRs in the majority of cases suggested selective pressure to
conserve functional structure by minimizing R events.39
However, the role of CDR3, which lies at the center of the
antigen-binding site,41 is difficult to assess in this type
of analysis, and VL is additionally important in antigen
recognition.42 Taking these findings together,
it appears that the final transformation event has occurred in a cell
with characteristics of an IgM+ memory cell.11
The absence of intraclonal heterogeneity suggests that the stage of
final transformation is postfollicular, which is, again, a feature
common to isotype-switched myeloma.18-20,22-24
Insight into the stage of arrest is provided from the analysis of
alternative isotype transcripts. In 2 of 4 IgM+ myeloma
cases, it was possible to identify CDR3-C1 and CDR3-C2 transcripts in each. These transcripts also displayed sequence identity
to tumor VDJ and intraclonal homogeneity. Although these tumors are
secreting high levels of IgM, cells within the clone appear to be
undergoing isotype switching to C. In contrast, although typical
myeloma cells are isotype-switched, there is evidence of bone
marrow-derived tumor cells producing VDJ-Cµ transcripts with complete
identity to tumor VDJ, as assessed from CDR2 or CDR3, and lack of any
intraclonal sequence variation.25-27 The nature of the
IgM+ precursor cell in conventional myeloma has not yet
been fully characterized, partly due to the very low numbers present,
but one possibilty is that IgM-secreting myeloma cells involve this precursor cell. The stage of transformation in IgM myeloma may immediately precede that in typical myeloma. It is also at this point
that the cell of origin in IgM+ WM is thought to
arise,43,44 but morphological and immunophenotypic features
suggest that this cell may be less mature.45 In addition, no isotype-switch variants were found in our analysis of 6 WM tumors
(Sahota SS, et al, manuscript in preparation).
The question arises as to whether the alternate isotypes originate from
the same cell or are the products of separate, clonally related
progeny. Evidence for separate populations was provided from our recent
study of cases of lymphoplasmacytoid lymphomas synthesizing clonally
related IgM and IgG.46 Divergent patterns of somatic
mutation in the 2 isotype transcripts in 2 of 5 cases proved that there
were 2 clonally related cell populations, 1 of which had switched
presumably by deletional recombination. This might suggest that, in
IgM-secreting myeloma, a few cells have undergone deletional switching
to IgG1 or IgG2. In contrast, in typical isotype-switched myeloma, the
majority have undergone isotype switch to a single constant region, but
there appear to be a few nonswitched cells and a few that have switched
to other constant regions.25-27 The fact that isotype
switching tends to occur on both alleles, both in mice47
and in IgG follicle center lymphoma, as shown by FISH
analysis,48 makes it unlikely that alternate isotypes
originate from the same cell by
trans-splicing.49
In 2 of the IgM MM patients, 1 allele is also involved in chromosomal
translocations, which in patient MOR was shown to map in the
JH region. Tumor-derived CDR3-C1 and -C2 transcripts were also identified in patient MOR; in this patient, alternate isotypes arising from the second allele by
trans-splicing are unlikely. However, contribution
by downstream CH regions on the functional allele could
occur, possibly by RNA processing. It is noteworthy that, in IgM
follicular lymphoma, such downstream elements also rearrange by
deletional recombination.48 Similar t(11;14q32)
translocations have been detected in 10% to 15% of typical
isotype-switched MM (Avet-Loiseau H, et al, manuscript submitted; reviewed in Hallek et al50).
Translocations involving chromosome 14q32 may furthermore be key events
in the pathogenesis of MM.50 However, it is evident that
not all of these events occur during the process of isotype switch, and
it would appear that switch recombination is not a prerequisite for
development of MM.
Location of the IgM-secreting tumors in the bone marrow supports the
concept that somatically mutated IgM+ B cells can leave the
germinal center, circulate in the blood,11 and move to the
marrow.51 The lack of ongoing mutations in these tumors and
the apparent sequence identity between IgM and IgG variants based on
CDR3-CH sequence would be consistent with isotype switch
occurring in a site where somatic mutation is silent, possibly in the
marrow. This clearly precludes the unmutated IgM-secreting plasma cell
of the primary response as the cell of origin of these tumors.12 Hypermutated IgM+ mature normal B
cells, isolated from the marrow, have been shown to secrete IgM when
cultured together with autologous bone marrow T cells and
T-cell-derived cytokines.51 These IgM+ cells,
which increase with age,52 could represent potential normal
precursors of IgM-secreting plasma cells. In normal bone marrow, there
are few identifiable IgM-secreting plasma cells, but IgM antibody
against tetanus toxin can be induced in bone marrow cells after
vaccination.53 Extensively mutated IgM+ plasma
cells have been found in normal gut mucosa, with the level of somatic
mutation comparable to IgA plasma cells at this site.54
Morphologically and phenotypically, these myelomas appear more mature
than the cells of WM, and they have evidently acquired the features
that lead to a clinical course of disease similar to typical MM. The
relationship of these tumors to the isotype-switched cells of
monoclonal gammopathy of undetermined significance (MGUS) is unclear.
It is rather surprising that the less mature IgM-positive cells lead to
clinical features indistinguishable from conventional myeloma, whereas
isotype-switched cells of MGUS do not. One possibility is that,
although MGUS may convert to MM in some cases,55 the route
to MGUS is distinct from that to MM. This suggestion is supported by
the finding of intraclonal variation, which is indicative of a residual
influence of the germinal center, in a subset of MGUS.31
Clearly, we need to probe further into the differences between these
tumors that have such impact on clinical outcome.
| |
FOOTNOTES |
Submitted December 11, 1998; accepted April 5, 1999.
Supported by The Leukaemia Research Fund, 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.
Address reprint requests to Surinder S. Sahota, PhD, Molecular
Immunology Group, Tenovus Laboratory, Southampton University Hospitals,
Tremona Road, Southampton SO16 6YD, UK; e-mail: sss1{at}soton.ac.uk.
| |
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TOP
ABSTRACT
INTRODUCTION
