Theodora Papadaki. Chrysouia Belessi. Xenophon Yataganas. Dimitra Anagnostou. Dimitris Loukopoulos. Somatic hypermutation of immunoglobulin variable ...
Christos Kosmas Kostas Stamatopoulos Theodora Papadaki Chrysouia Belessi Xenophon Yataganas Dimitra Anagnostou Dimitris Loukopoulos
Somatic hypermutation of immunoglobulin variable region genes: focus on follicular lymphoma and multiple myeloma
Authors' addresses
Summary: Atialysis ol the rearranged itmnunoglobulin variable region gene hypermutation has provided important information concerning the clonal history and oiHogenetic origin of various B-cell lymphoproliferative disorders. Under the selective pressure of antigen, mutational events in imtiuinoglobulin genes will ftiie tune survival of B-cell cloties bearing immunoglobulin with high affinity for antigen. Our sttidies aimed at analyzing neoplastic disorders originating frotn gertnina! and post-germinal center B-cells: follicular lymphotna and tnultiple tnyelotna. respectively. Despite the already acknowledged evidence for a selectable distribution of mutations within the clonal immnnoglobnlin variable heavy chain genes, very little is known about the contribution of light chains in the process of antigen selection. In foilicuiar lymphoma. a more limited pattern of somatic tnutation witli less evidence of antigen selection was observed in variable K light chain genes (40%) than in their partner heavy chain geties (80%). In myeloma, hypermutation of variable light chain genes, with a distribution suggestive of antigen selection, was frequently observed. Based on these data and recent reports il appears that the hght chain expressed by the cionogetiic myeloma B-cells plays a pivotal role in the atitigen selection process. Additionally, abortive K light chain variable region genes in X-expressing myelomas carried a significant number of somatic mutations indicating tlut the cell of origin is open to the hypermutation machinery at that particular developmental stage irrespective of antigen selection.
CKriilos Ko,smas'. Ko^tas Stumatopouios', Theodora Papndoki', Chrysouia Belessi', Xenophon Yataganas', Dimitra Anaqmslou^, Diiniuis Loukopoulos'. 'Eirsi Department of Medicine. University of Athens School of Medicine. Laikon General Hospital, Athens. Greece, 'Hemopathology Unit. Evangelismos Hospital. Athens, Greece. Correspondence to: Chris (OS Ko,?mas First Department of Medicine University of Athens School of Medicine 21 Apolloniou Street 163 41 Athens. Greece Fax: 30 1 I 301 9 9 6 2917 Acknowledgemenls
The atithors thank Mrs Stavroula Afeiidaki and Evi Pouliou. Hemopathology Laboratory, Evangehsmos Hospital. Athens. Greece, as well as Panagiotis Kyriazopoitlos and Pana^iota Avraniopoulou, First Department of Medicine, Athens Utiiversity School of Medicine, Laikon General Hospital, Acheiis. Greece, for expert technical assistance, Dimitris Loukopoulos was supported by the RND Program STRIDE No. 66 (Secretarial for Research and Technology),
Introduction Ontogeny within the B-lymphocyte hneage consists of an antigen-independent and an antigen-dependent phase. The antigenindependent phase takes place in the bone marrow and is characterized hy an ordered process of immunoglobuhn (Ig) gene rearrangements leading to the assembly of distinct variable (V), diversity (D) (for heavy chains (HC) only) and joining (J) gene segments; this phenomenon is known as V(D)J recombination (1, 2), Snccessfiil rearrangement of HC Ig genes and subsequently that of Ught chain (LC) Ig genes (K or X) will enable tlie developing B cell to express later on its surface a fully functional
Immunological Reviews 1998
Vol. 162:281-292 Printed in Denmark. AH rights reserved
Ig recepior with unmutated V-region sequences. The antigendependent phase will start when the "tiaive" B cell exiting the bone marrow compartment enters into the follicles of the sec-
CopjTighi © Mtmksgaard 1998
Immunological Reviews ISSN 010,5-2896
ondary lymphoid organs, the natural anatomic place where germinal centers (GCs) are formed, GCs are formed after antigenic
281
Kasmas et al • Hypeimutation ofV genes in FL and MM
stitnuladon throngh a T-cell-dependent tnechanism; it has been postulated that ihey represent the siies responsible for both the affmity maturation of aniibodies and the formation of B-cell memory (3), The contact of a naive B cell newly entering the GC tlirough a low-affmity surface Ig with antigen will lead to a high proliferation rate associated with somatic hypermutation of Ig genes, a process characterized by mutations within V regions at an estimated rate of 10"^ to lOVbp per generation (4), Somatic hypermutation of Ig genes concerns their V region and is the lialltnark of antigen selection, a process diat will tum a GC naive B cell expressing a low affinity slg to a high-affinity antibody producer as well as a long lived recirculatitig tnemory B cell. Antigen selection, seen as a dynamic process, is an ongoing phenomenon in the GC that will eventually lead to the survival of B cells expressing higli-affmity Ig V-gene mutants; in contrast. B-cell clones with less affinity for antigen are destined to undergo apoptosis (4), When antigen selection is accomphsbed, the hypermutation process ceases; typically, memory B cells and plasma cell precursors are not accttmulating any fnrther hypermutations in their variable heavy chain (Vn) and variahle light chain (V,) genes.
Follicular lymphotna (FL) is a low-grade B-cell malignancy most likely deriving from the neoplastic transformation of GC B celis. Il has convincingly been shown that the growth pattern of FL cells is determined by the signaling function of their sigs (i 1). FL cells carry sIgs whose HC V regions exhibit ntimerous mutations with a tendency to cluster in ihe CDRs. a fmding indicative of selection of the neoplastic clone by antigen, as well as a pattern of ongoing accumulation of CDR-Iocated mutations when studied at different phases of disease evolution (12, 13).
Clustering of mutations occurs predominantly within the complementarity determining regions (CDRs: CDRl, CDR2 and CDR3) ofthe respective polypeptide's V region (5); the explanation given for this phenomenon in inilial studies was rather teleologic, namely that affinity-hased selection should enrich for B cells carrying somatic mutations in CDRs, the regions responsible for antigen binding (6). However, subsequent experimental data have pointed out that targeting of sotnatic hypermutation in CDRs is indeed an intrinsic property of this mechanism (7-9). Interestingly, separation of the intrinsic properties of the somatic hypermutation process from the effects of antigen-driven selection has been achieved in an experimental model where V,, genes, open to accumulate mutations but not selected for affmity and carried as "passenger" transgenes not contributing to an antigen-specific immune response (7. 8). exhibited a very strong intrinsic hypermutation hotspot in CDR 1,
Multiple myeloma (MM) is a malignancy of the immune systern characterized by the presetice of a cotitinuously differentiating population of mainly late-stage B cells giving rise to plasma cells (14). The analysis of VH-region gene rearrangements in MM indicates that, before transformation, the malignant stem cell (whose exact origin remains elusive) has already undergone antigen selection with consistent lack of intraclonal diversification (15. 16). Analysis of LC V-region genes has revealed somatic hypermutation of almost tlie same magnitude as has heeti observed for VH genes (17-19), This fmding is in contrast to our observations in FL V,, genes and offers more direct evidence that MM originates from transformation ol late post-GC B-cell clones. Furthermore, it indicates that hypermutation of VL genes might serve as a surrogate marker of discrete developmental stages regarding GC and posl-GC B-cell development.
Analysis of V-region mutations can provide important information regarding the ontogenetic stage of various B-cell populations. Moreover, analysis of V-gene mutations in B-cell lymplioprohferative disorders has helped to trace the developmental stage at which neoplastic transformation has taken place and assign these cells to their correspondijig normal counterparts (1 0), In addition, this type of analysis has pointed out the correlation hetween microenvironmental localization and fnnctional properties of particular B-cell populations pertinent to the developmental pathways outside and inside GCs. 282
Immunologiea! Review.s 162/1998
We have examined the extent of somatic hypermutation in FL V,, LC genes and compared their mutation profile to that of HC V genes of the same lymphoma cases in order to obtain evidence about the putative importance of FL cells" K LCS in the process of selection by antigen. We have demonstrated [hat despite the fact that nitLtations do occnr within V,, genes of FL, their potential contrihntion to the process of antigen selection appears to be less important than that of VH genes. Given that FL derives from the neoplastic transformatioti of GC B cells, it is possible that at discrete stages of this developmental phase the hypertnutation process for V^ genes is silent or less active.
It is anticipated that studying somacic diversification of V genes in lymphoprohferative diseirders such as FL and MM would provide information regarding i) the potential temporal relationship between neoplastic transformation and antigen selection, ii) possible biased V-gene repertoire usage by neoplastic B cells, iii) tbe contribution of V, to the antigen selection process and iv) a better understanding and more direct evidence about the postulated assignment of these B-cell malignacies to the respective GC and post-GC stages of B-lymphocyte differentiation.
i
Kosmas et al • Hypermutation of V genes in FL and MM
DPW:
Hpl,
Fig. 1. Amino acid and nucleotide sequences of VH genes from FLs. Each V^ or V^, sequence is compared witli the most ciosely matching germiine gene. Sequence identity is indicated by a dash and amino acid differences are shown by upper case letters (R mutations) or lower case letters (S mutations), Mutations in J,, or J^ genes are underlined. The CDR and FWR assignmeiiL is according to data from Kabat et al. (23) and nomenclature of V,, genes at;tording to Tomlinson et ai, (24), Assignment of D and JH gene segments was performed according to Yamada et al, (34), The numbering and nomenclature of V^, genes is according to Klein et a!. (28), in sequence FL6-K, asterisks denote a deletion of 6 bases in FWR3,
ESOGflLvopflceLiaacMScnTs S T * ™ HV
EVQLVE ^ L V a P « = a L K = « ^ G F T T S
I
.T««W
II
•
EVQLV
ri
E5SSBT1*1'JU>5VKC
1—P-g
•
1— • - • m -V
—1^—TU-tf
RDWCKLEWVA V
qv— — i—^r-a—tl- —
V3-J0.3
OVQLV
iCYapi- a nlie. KVMJHPG^GI,!IWIG
i
ilsPSFQC OVTISHWSISIRVLO
iraSUWTDTAHUCM
F»-OP CDu
—i
,
-
1
^
—1
™^ FSSLSAGVQDftVTlTC
LLI'T
MSOOI:
«-
rL!-«:
,^—F—
5
ITTCQGTPLKlKfi
teevEASVCDBVTlIC
ssmj.
HYMUPGUKPh
AAESL06
SVrSETECaGaCTDFTlTlSSIareoriLmc
COB TqoOTmiJllK
Ig genes in follicular lymphoma
The material for the present study derived frotn fresh frozen lymph node specimens from 14 FL patients at diagnosis. Clonal VH and V^-gene rearrangements were amplified by PCR from i 0 FL cases and sequences analyzed by methodology described in previous reports (20-22), The numbering and nomenclature of VH and V., segments was carried out according to previous reports (23-25), Nucleotide sequence data comparisons were performed with the corresponding germline sequences using the GenBank/EMBL database. Statistical analysis ofthe distribution of tnutations was carried out by the binomial probability model of Schlomchik et al, (26) as modified by Chang & Casali (27). Each codon was assessed separately for the presence of somatic mutations. When more than one nucleotide substitution was encountered within a three-hase pair codon sequence, it was postulated that mutations were introduced sequentially
J,i
from the first through the third nucleotide in order to be assigned as being of the replacement (R mutation: nucleotide change leading to amino acid substitution in the encoded polypeptide) or silent (S mutation: nucleotide change leaving the encoded polypeptide unaltered) type (16). Distribution of rearranged V, (and V^ genes in FL clones On the basis of homoiogy to the closest germline (unmutated) Vji and V^ sequences, the V^ genes used by the FL clones analyzed in the present study were found to correspond to germline VH genes of the VH3, VH4 and VnS-gene families (Table 1, Fig, 1), while the rearranged V^ genes were found to bear homoiogy to 6 different germline V^ genes (V1, Vb, Vd, O2-12, O8-18, VJV) (Table I, Fig. 1) (28). Restricted or hiased utilisation of V-region genes is observed in some human B-cell tumors (29-32). In contrast, FL neoplastic cells carry sIgs of an tinbiased V,, repertoire (33),
Table I. Vn and V^- -family assignment and sequence homoiogy to the closest germline gene FL clone
Closest germline VH gene
Homoiogy to germline
Closest germline V^ gene
Homolog/ to germline
FL1
f1-p1
95,2
Vb
94,0
(VH3)
FL2
DP-58 (VH3)
89.7
Vd
97,1
FL3
V4-34 (VH4)
88.7
V..-IV
97,8
FL4
f1-p1
96.3
Vd
95,1
FLS
V3-23 (VJ)
87.4
O2-12
99,6
FL6
V5-51
(VH5)
88,8
Vd
93,3
FL7
V3-43 (VH3)
89.8
O2-12
98,5
FLS
V3-30.3(Vi3)
87.4
O8-18
91.2
FL9
DP-59 (VH3)
93.9
V1
95,9
FL10
V3-48 (V,3)
90,1
oa-18
90.9
(VH3)
Immunoiouicfll Reviews 162/1998
283
Kosmas et al • Hypermutation of V genes in FL and MM
with a frequency of expression of particular Vn-gene family members closely similar to the one reported for mjrmal periph-
Tflble 2. Closest germline D and JH genes used by the HC Ig ofthe FL clones
eral blood lympliocytes (34), CDR3 formation in HC and K LC V regions of individual FL clones
HC genes
FL clone
D gene used
JH gene used
FL1
DKf
jK3b
FL2
DXP'1
Jntb
FL3
ND
ND
FL4
DK4
|H5b jM4b
FL5
LR4
The FL clones analyzed exhibited preferential utilization of the
FL6
DIR
JH4 gene (Table 2) in accordance with what has been described
FL7
D21/9
jH5a
for normal peripheral blood B cells (34), D segments used by
FL8
DXP4
)H6b
the individual FL clonogenic B cells are showai (Table 2), The
FL9
DAI
lH4b
nomenclature and comparisons to germline D segments was
FL10
DHQ52
jM3b
carried out according to Yamada et al, (35).
ND: not determined
K LC genes At the level of individual J^-gene segments used by R clones
model, considering that each VH and Vi-gene sequence is
that we analyzed, an almost equal representation of J. 1, ]^1, JK4,
(Rf) for certain V genes are inherently higher than expected in
and ],,5 genes was observed (Fig. 1), with no similar hias to that
CDRs as computed according to the iniierent ability of their
of the preferential JH4 gene usage by normal peripheral blood
codons to mutate (27).
assessed codon by codon and that the R mutation frequencies
and FL B cells. N-nncleotide insertions were detected in 4 out
The binomial model proposes that the distribution of
of 10 clones (Fig. I); furthermore, the length of tlie identified
mutations in the CDRs and FWRs - provided mntations occur
N segments was short (3 nucleotides in all cases; the number
hy chance - depends on: 1) the relative sizes of these regions
of inserted N nucleotides should always be a factor of 3, other-
(0.25 for the CDRs and 0,7 S for the FWRs); ii) the assumption
wise, the V,,-J,,- junction will he rendered out-of-frame).
that the ratio of R to S in a protein region not under functional
The numher of inserted N nucleotides at the V^-J^ junctions
constraint should be close to 2,9 {however this ratio should
was much smaller than that in the corresponding VH-N-DH
change if analysis is carried out according to the proposals of
junctions. This fmding is in accordance with what happens in
Chang & Casali, which take into account the inherent tendency
normal hone marrow or post-bone marrow mature B cells; the
of certain codons, particularly those of CDRI, to mutate with a
scarcity of N nucleotides at the V^-J.. junctions is thought to
higher frequency irrespectively of antigen selection), and iii)
result from a progressive drop in the activity of TdT at the post-
the assumption that twice as many R mutations occur in the
|i-HC expressing pre-B-cell stage (36), TdT is the enzyme
FWRs. since some of these having deleterious effects will be
responsible for non-templated nucleotide addition (N-region
lost from a clone of antibody-producing cells and will not he
diversity) at the V-D and D-J junctional boundaries of Ig HC.
included in the total number of observed R mutations in FWRs.
Ehiich et al. (37) demonstrated that K LC gene rearrangement
Therefore, the total nnmber of V-region mutations would be
can take place in mice unahie to rearrange Ig HC genes,
distributed as follows: N = RCDR + S + IK^-WK- Therefore the
although with lower efficiency, at the pre-B cell stage, where
prohability (p) for k"R mutations to occur in the CDRs for a
TdT is active. It is possihie that N regions seen in the V,- rear-
total nnmber ofmutations equal to N is calculated by the equa-
rangements analyzed are the result of TdT activity in cells
tion p - [N!/k!(N-k)!]q'-(]-(i)^^ where q is the chance for an R
undergoing early K LC gene rearrangement.
mutation to occur in CDRs,
Somatic hypermutation - distribution of mLrtations in the VH and V^ LC-region genes
In order to evaluate whether the number of R in the CDRs was significantly higher than the number expected randomly, we applied the binomial probability model adopted by Shlomchik et al. (26), taking into account the relative sizes of the CDRs and framework regions (FWRs) in V,i, V,,. and V^, The method of Chang & Casali (27) was apphed in order to modify the above
284
lmiiiunoloj]ii,-ijl Reviews 1 6 2 / 1 9 9 8
HC genes All cases demonstrated extensively mutated Vn-gene sequences. Mutations in the rearranged VH genes clustered preferentially in the CDRs (Fig. i ) . Most of the mutations occurring in FWRs were S; in contrast, mntations observed in the CDKs were nsually of the R type. The R:S ratios in the CDRs were significantiy greater than 2.9 and, taken together with the R:S ratios in the FWRs, the distribution ofmutations differed significantly from
Kosmas ei al • Hypermulation of V genes in FL and MM
Table i. Distribution of mutations in the Vy region of FL cases
about the effects of somatic hypermutation on VL-region genes in affinity maturation ofthe antibody response, FL comprises a
R/S.
group of lymphoproliferative disorders with varying histo-
FL clone
Observed
Expeaed
Observed
Expected
P
FL1
2/1
2/0
2/7
7/3
0,23
FQ
11/5
S/1
3/11
17/7
0,009
FL3
11/3
6/1
8/12
19/8
0,045
FL4
3/1
2/0
2/6
7/3
0,19
immunohistocliemical parameters were examined: ihe mor-
FLS
15/2
7/2
8/18
25/9
0.002
pholtigical pattern (nodnlar versus diffuse), liistologic grade
FU6
10/4
5/1
8/10
20/6
0.052
and bcl-2 protein, Ki-67 and CALLA antigen expression. As
FL7
11/2
5/1
6/11
17/7
0,016
expected, cases differed as regards the above parameters. No
Fie
H/4
6/2
8/11
23/6
0.009
correlation was identified between any of the above parameters
FL9
9/3
3/1
3/3
10/4
0,003
FL10
10/3
5/1
7/11
18/7
0.042
paihological and clinical behaviour (40). In an attempt to correlate the moIecLilar fuidings with the histological profile ofthe FL cases that we analyzed, a number of histopathological/
and the frequency ofmutations either in the Vn (3r V^ genes. It is not known whether the V., genes expressed by the FL clones have any role in the antigen selection process or simply
that predicted by die modified binomial model (27), if muta-
constitute "innocent bystanders", just supporting the critical
tions had been iiilroduced randomly (Table 3).
conformational changes occurring in the VH region ofthe anti-
All existing studies concerning FL slg Vn genes point t(j the
gen-recognizing slg.
fact that these genes are affected hy the somatic hypermntation
The relative scarcity or complete ahsence of mtuations
machinery, with mutations exhibiting a strong tendency to
within certain V,- genes nsed by FL clones might well he attrib-
cluster in the CDRs; these observations are suggestive of selec-
uted to the inherent composition of codons of the genomic V^.
tion ofthe neoplastic clone by antigen (1 1-13. 38), although
locus. which is predicted to accumulate mutations less fre-
exceptions to this rnle have heen reported (39), hi the present
quently than V,, genes, as originally calculated in the model
study, the results concerning somatic hypermutation of the
proposed by Chang & Casah (27). In this context, somatically
rearranged VH genes imply that in all the FL cases analyzed, the
mutated rearranged V,, genes tuider the selective pressure of
clonogenic malignant GC B-cells were affected by antigen
antigen tnight pair with relatively nnmutated V^ genes and pro-
selection, which operated on and changed the conformation of
vide the FL B ceil with a selectable high-affinity slg. A similar
their slg HCs, by means of favoring the snrvival of high-affinity
situation has been convincingly exemplified in the case of
mutants.
Hodgkin's disease (HD)-Reed-Sternberg (HRS) ceils (41).
K LC genes In contrast to VH genes, the pattern ofmutations ofthe V^ genes analyzed in the present study was not uniform. Specifically: i) in case FL4 (with tiie closest homologous V, gene being Vd), the R:S mutation ratios in the CDRs were signilicantly greater
The fact that, in a large percentage ofthe cases under study (50%), V,- genes were not participating in or affected hy the aJitigen selection process leads to tlie idea that in some B-cell clones selection may simply require affinity-improving mutations in Vn-region genes only. It is possible that the recognition
than 2,9 and the distrihution of mtitations differed significantly from that expected to have occurred by chance according to the
Table 4. Distribution ofmutations in the V^ region of FL cases
probability calculated hy the binomial model (P=O,OO25; R/S.
Table 4); ii) in cases FLI, FL8, FL9 and FLiO. mutations ofthe R type, when encountered, were fewer in number but demonstrated a tendency to cluster in the CDRs, R:S mutation ratios
R/S,F
FL clone
Observed
Expected
Observed
Expected
P
ai
9/5
4/1
0/3
9/3
0.0005
FL2
3/0
2/0
0/5
4/2
0,172
were greater than 2.9; furthermore, a tendency was observed
FL3
2/1
2/0
0/5
4/2
0,79
for S mutations to distribute in FWRs; iii) in the remaining
FL4
7/2
3/1
0/5
7/3
0,0025
cases (FL2. FL3, FL5, FL6 and FL7), either no R mutations were
FL3
0/0
0/0
0/1
0/1
"(NS)
observed (FL3 and FL5) or their distribution was similar to the
FL6
4/4
4/1
3/8
10/4
0.15
FL7
0/1
1/0
0/3
2/1
«(NS)
FL8
6/3
3/1
0/6
a/3
0.01
FL9
6/3
3/1
1/4
7/3
0,047
FLIO
11/7
5/1
0/7
13/6
0,006
one predicted to occur hy chance (Table4, Fig. i). For cognate antigen recognition, it is postulated tliat CDRs from both V^ and V,. contribute to the formation ofthe antigenbinding cleft of a B-cell slg molecule. However, Httle is biown
NS: not significant
Immunoiogicai Rcviewi 162/1998
285
Kosmas et al • Hypermutation of V genes in FL and MM
of certain antigenic epitopes is critically dependent on confor-
dard methods as already described in our previous report (18),
mational changes provided by the CDRs ofthe VH region alone.
On the hasis of serum immunoelectrophoresis/immunoblot
Apart from FL, another B-cell neoplasm that has recently
analysis, 9 cases were assigned as K and 6 as >.-expressing MM,
been ontogenetically Icjcated to the GC is HD (41-43). Elegant
while 2 cases were non-secretory MM. Rearranged clonogenic
studies at the single-cell (HRS-cell) level have provided compel-
V^, and V;,-gene sequences were amplified and analyzed accord-
ling evidence that VH genes carry a high number of somatic
ing to standard methodology as described in our previous stud-
mntations with a distribution strongly suggestive of antigen
ies (18, 22) and hy other investigators (20, 21), Seqnence anal-
selection, whereas in certain cases an ongoing pattern ofmuta-
ysis, gene assignment and statistical analysis of mutations for
tions is ohserved. However, in several instances, in-frame
clonogenic MM V,^ or Vj. genes were carried out as described.
VH-gene rearrangements have been rendered non-functional by
For V, genes the nomenclature and codon numbering was
stop codons as a result of point mutations, or deletions of base
applied as proposed by Chuchana et al, (44) and Williams &
stretches within the V^-gene sequence in others. This holds true
Winter (45),
in cases of classic HD (nodular sclerosis and mixed cellularity
In many of the cases that we analyzed, the distrihution of
subtypes), whereas recent studies in lymphocyte-predominant
mutations in the CDRs was significantly higher than expected
HD have revealed that VH genes in these cases exhihit a high
hy this model and, correspondingly, the same ratio in the FWRs
degree of ongoing somatic hypermutation compatible with
was significantly lower than expected. According to the bino-
snstained antigen-driven selection, similar to the pattern of FL,
mial probability model and taking into account its modification
More importanliy, these mutatiom do not appear to disable the
by Chang & Casali (27), namely that an inherent liigher-than-
VH-gene sequence (42, 43). Therefore, the neoplastic GC B cells
expected mutation frequency is ohserved in CDR I in the
in classic HD are able to survive, at leasi in part, in the absence
ahsence of antigen selection, significant clustering of R mnta-
of antigen selection, raising the intriguing possibility of the
tions in CDRs occurred in 6/11 (55%) MM V, genes and 2/6
existence of a potent transforming event maintaining their
(33%) V^ genes.
growth and expansion. In our analysis, only a single FL clone was found to carry an otherwise in-frame trnncated V^ gene
Sequence analysis of MM V, and V^ genes
with a 6-bp deletion in FWR3 (Case FL6-K; Fig. 1), which
RT-PCR analysis identified V,,. LC mRNAs in 11/17 cases
might be potentially functional as a result of an even loss of
included in the present study; 9/1 1 were K-expressing MM,
bases that should give rise to an expressed slightly shortened V^
whereas 2/11 were non-secretory MM. Yy, LC mRNAs were
protein (immunohistochemistry positive for K LC expression).
identified in 6/17 cases in the present study
Similarly to otir results, other investigators have never reported trnncations. out-of-frame rearrangements or stop codons in FL-rearranged VH genes. These data, in contrast to those ohtained in HD, might provide clues ahout understanding dis-
Paraffin-embedded, trephine bone biopsies from the 2 cases of non-secretory myeloma were suhjected to immtmohistochemical analysis for the presence of cytoplasmic Ig chains. Both cases were positive for cytoplasmic K LC.
crete stages of intra-GC B-cell development associated with differential qualitative and quantitative requirements for antigen selection. In this line of evidence, the HRS cell might correspond to the transformation of an early centrohlast entering the dark zone of ilie GC, where a high proliferation rate coupled with a high frequency of somatic mutation witiiin the VH region are associated with an increased possibility of deleterious Vii defects that would ultimately lead the normal B-cell to death. In contrast, FL cells correspond to late centroblast or centrocyte GC B-cells with a pattern of mntations permissible for eel! survival by ongoing rounds of antigen selection,
K dnd V^-gene usage On the basis of homoiogy to germline (nnmutated) V^ sequences, the V,, genes used hy the myeloma clones analyzed in the present study were fotmd to bear homoiogy to 6 different germline \ genes (O8-18, Vh, VI, O2-12, V^-IV, Ll 1; Table5) (28), Analysis of V^ genes in six MM cases identified equal distribution between VJ (DPLl, DPL4), VJl (DPL12), Vxlll (DPL16, DPL23) and V,VI (IGLV6SI) (see also Table 5) gene famihes with homoiogy to these 6 germline V,, genes (45). Despite the fact that the total numher of cases analyzed does not permit statistical evalnatioii (jf the results concerning V^-family gene nsage, the rearranged V^ genes identified in our
Ig LC genes in multiple myeloma
Bone uiarrow aspirates as well as trephine hone biopsies were obtained from 1 7 patients with overt MM and analyzed by stan-
286
ImmunoiogicQJ Reviews 162/1998
study (18) and three other reports (17, 19. 46) were compared for differences and similarities regarding individual gene usage hy MM clonogenic B-cells (Tabie 6), It can he seen that VJ-gene
Kosmas et al • Hypermutation of V genes in FL and MM
Table S. V^ and Vj-gene assignment and sequence homoiogy to the closest germline Myeloma clone/ RNA sample
Closest germline ^n- or Vx gene
or Vl gene family
VK
Homology to germline (%)
MM2-K
Vb 02-12
VK-I
80,0
MM3-K
VK-IV
VK-IV
94,8
MM1-K
MM4-K
02-12
VK-I
VK-I
92,3
94.8
MM5-K
L11
VK-I
MM6-if
VI
VK-I
87,5
MM7-K
Vb
VK-I
93,1
MM8-K
VI
VK-I
86.6
MM9-1C
VK-IV
VK-IV
86,7
MM10-K
VI
VK-I
81,0
MMII-K
O8-18
Vv-I
97.0
89.4
Thble 6. Collective data on V.-gene usage in MM from various reports
O8-18
02-12
L11
1/4(25%)
1/4 (25%)
1/4 (25%)
0/4
0/4
17"
2/3 (67%)
0/3
0/3
1/3 (33%)
0/3
46
0/11
18
A27
VK-IV
1/11 (9%)
2/11 (18%)
1/11 (9%)
2/11 (18%)
4/9 (44%)
0/9
0/9
0/9
8/27 (30%)
3/27(11%)
2/27 (7%)
3/30 (10%)
3/9 (33%)
Reference
19
3/27(11%)
' Only functional in-frame V^ genes are considered from this study
(FWR2), characterized hy replacement of Gin hy His (Fig. 2), Analysis of V>. genes revealed significant clustering ofmutations
MM12-?.
DPL23
V?.-lll
82,1
within CDRs in 2/6 (33%) cases. In 2 cases (MM12->. and
MM13-?.
DPL4
VX-I
93,9
MM I 5-X,), a heavy mutation load was observed in Vj. genes with
MMH-X
DPLl
VX-I
90,3
significant clustering in the former and a trend to significance
MM15-^
DPLl 2
VX-II
90.2
for antigen selection in the latter, providing fnrther evidence
MM16-X
DPL16
VX-IN
91,6
regarding the heterogeneity of individnal MM clonogenic
MM17-X
IGLV6S1
VX-VI
96,3
B-cells (Table 7). In total, when both V,. and V^ genes nsed by MM cases in onr stndy are concerned, 8/17 (47%) demonstrated an R/S mutation distribution being compatihie with the notion
family members (08-18, O2-12, L i l ) are utilized more fre-
that the clonogenic B-cell precursors of MM have been selected
qtiendy than olliers in MM, but with similar frequencies to
by antigen (Table 7). In 2 cases (MM2-Kand MMI 5->.), the dis-
normal peripheral hlood, fetal bone marrow and lymphopro-
trihution ofmutations showed a trend to significance (P valnes
liferative disorder B cells (47),
of 0.062 and 0,061, respectively), thus demonstrating a ten-
N-nucleotide insertions were detected in 5 out of 11 MM clones in the V^-J,. junctions; furthermore, the identified N seg-
dency towards antigen selection (Table 7). MM is characterized by the clonal expansion of B cells at
ments were short (3 nucleotides in 4 cases and 6 in 1 case). In
the preplasma and plasma cell stage. Various populations of
contrast, no N-region diversity was ohserved in V^-J^ junctions.
pre-B and B cells in the peripheral blood are clonally related to
Functional V,-J,_ rearrangements do not have N-nucleotide
the tnyeloma clone in the bone marrow (48-50), Seqtience
insertions at the junction or deletions in the Jx-gene segment
analysis of (he VH genes in MM shows that extensive somaiic
(44),
mutations occur in the conrse of the disease, clustering in the CDR regions; the majority of these mutations lead to amino
Somatic hypermutation - distribution ofmutations in V^ and
acid replacements. This finding imphes that the myeloma cefl
V^-gene sequences
precursor has undergone antigen selection via its surface Ig
Many cases ( 6 / 1 1 ; 55%) demonstrated extensively mutated V,-gene seqnences. The pattern of mntations in the V^ rear-
molecules (15). V,, or Vj. LC contrihnte to ihe three-dimension conforma-
ranged genes shows an asymmetric distribution in CDRs com-
tional orientation of the antigen-binding site. It is therefore
pared to FWRs. However, most of the mutations occuring in
important to examine whether aniigen selection, apart from
FWRs were S, whereas mutations in the CDRs were more fre-
driving the somatic hypermntation process operating on the VH quently ofthe R type (Fig, 2, Table 7). Analysis ofmutations was genes, by means of allowing high-affinity mutant-bearing carried out codon hy codon. It is noteworthy that, as in other B cells to survive, requires hypermutation ofthe V,, or V^ LC studies. Ser residues represent mutational "hotspots" in many ofthe cases analyzed (Fig, 2). For instance, Seri 1 has been iden-
genes expressed by the selected B-cell clone. Extensive somatic mutations of the V^ or V,, genes rear-
tified as a mntational hotspot in CDRI both in the present and
ranged in K or X LC-expressing MM, respectively, were ohserved
in other studies (7, 17). In both V^-IV genes identified in our
in many cases in the present study. R mutations tended to clus-
analysis, a non-conservative change (G-^C transversion leading
ter preferentially in the CDRs, usually giving R:S mntation
to CAG-*CAC; Gln-»His) occurs in amino acid position 38
ratios significantly higher than the ratios predicted by the binoImmunolojjical Review,s 162/1998
287
Kosmas et al • Hypermutation of V genes in FL and MM
Fig. 2. Ammo acid and nucleotide sequences of V^ and V, genes from K or ?t LC-expressing myelomas respectively, Each V^ or V, sequence is compared with the mo-ii closely mattliing germline geac. Sequence identity is indicated by a dash and amiiiu acid differences are shown by upper case letters (R miuations) or lower case letters (S mutations), Muiation.s in J, genes are underlined. The CDR-FWR assignment is according to data from Kabat ttt al, (23), while V, and V^ gene nomenclatures are according to Klein et al, (28) and Williams & Winter (4,'j) respectively.
mial model if these had followed a random distribution. This high R:S muiation ratio in the CDRs implies thai, in a large nxunher of these K or ?. LC-expressing MM cases, the somatic hypermutation machinery for the V, geties has operated at the level of the myeloma B-cell precursors and those that were carrying mutations suitable for antigen selection have undergone suhsequeni clonal expansion. Previous studies into B-cell chronic l)aiiphocytic leukemia (B-CLL) have shown that V,genes show very Hmited or no somatic mutations when compared to their respective germline (51). In 5 cases from our study, mutations were scattered throughout the V^, region with
Mile 7. Distribution ofmutations in the V,,- and V^^-region genes of MM
Myeloma clone/
FvSjCDR;
RNA sample
Observed
Expected
Observed
Expeaed
P
MMI-K
9/3
3/1
0/3
8/3
0,0013
MM2-K
17/5
10/3
15/9
25/8
0,062
MM3-K
4/1
4/1
2/5
7/2
0,20
MM4-K
3/1
3/1
1/7
6/2
0.19
MM5-K
9/3
6/2
2/14
15/5
0.012
MM6-K
9/3
6/2
3/12
15/5
0.012
MM7-K;
12/2
3/1
0/1
8/3
0,0000
MM8-K
13/7
9/2
1/17
20/7
0,047
MM9-K
8/3
9/2
5/22
21/6
0,934
MM10-K
13/6
10/3
9/18
25/B
0.119
MMII-K
4/1
2/0
2/1
4/2
0.015
MM12-X
29/9
18/5
19/23
43/14
0.017
MM13-?.
4/1
4/1
1/10
8/3
0,22
MM14-X
12/2
8/2
6/16
18/8
0,041
MM15-X
12/4
7/2
9/8
17/7
0,061
MM16->.
6/4
6/2
6/13
15/6
0.136
MM17-?.
S/2
3/0
2/2
6/2
0.102
288
Immunoiogicul Reviews 162/1998
no asymmetry in the distrihution of R mutations in favor ofthe CDRs. This finding is not consistent with the operation of an antigen-driven process, raising the possibility that in certain Igexpressing mature B cells - die normal counterparts ofthe clonogenic cells of certain late B-cell lymphoproliferative disorders — it is sufficient for the antigen selection mechanism to operate at the level ofthe Vn region. Alternatively, it may imply that the neoplastic event occurred before selection by the antigen had taken place and was potent enough to prevent apoptosis in the absence of antigen selection. The above findings support the notion that the nature ofthe myeloma B-ceil progenitors is heterogeneous. Somatic hyperniLitation in non-secretory MM The 2 cases of non-secretory MM in our series {MM4-K and MM6-K:) had evidence of K LC-gene transcription, as determined by RT-PCR, and iniracytoplasmic K LC expression, as assessed by immunohistochemistry Both myeloma clones carried functional V,- genes with R:S mutation ratios in the CDRs of 3:1, not differing from the raiio predicted by the binomial model, although, in boih cases, the distribution of tinitations in FWRs was significantly different to the one expected if mutations were due solely to chance. However, MM6 exhibited a distribution of mutations throughout the V.-gene sequence strongly suggestive of being selected by antigen (Table 7). One of these cases lacked detectable intracytoplasmic y HC by immunoperoxidase but was HC(+) by RT-PCR. The absence of extensive somatic mutations in the CDRs of the V,, genes used by the non-secretory myelomas analyzed in our study would suggest that possible defects in the expression ofthe encoded K LC prevexited the B-cell precursor of die myeloma clone from undergoing antigen selection.
Kosmas et al • Hypermutation of V genes in FL and MM
the mechanism targeting somatic mutations can provide important information relevant to the ititra-GC versus post-GC developmental B-cell stage of HD and MM respectively: In X LC-expressing B cells, non-function a! V^-gene rearrangements may remain in the genome or be deleted (53), Rassenii et al. (52) described three possible mechanisms of inefJT OfS tar oTT MC
fective V,,-gene rearrangements in X LC-expressing B-CLL: i) »: w A oi ,S; ,;.
occurrence of frameshift mutations in the V.-J,. junction, apparently introduced during die process of Ig gene rearrangement; ii) in some cases, direct rearrangement of V, genes to Kde, a
Fig. i. Sequences of abortively rearranged V^ genesft-omX LC-expressing myelomas. Above each nucleoiide sequence ilit dedut:ed arniiio acid "sequence is Liidicaiud, ,'\min() acid inimlitTing and CDR position is according to Kabat ci al, (23) anc! Klein ci ai. (28), 8SK, Sdv and 92K represent ilie numbers of genomic DN.A samples corresponding to MM I 6-?., MM I 3->. and MM 12-/. respectively.
DNA element positioned 2+ kb 3' to the C, region, rearranging with the V;,, or J^ region in order lo eliminate the C-region exon (54), and iii) introduction of a stop codon in the open reading frame. It seems that a mechanism similar to tiie third, mentioned above, operated
in case 92K (corresponding
to
MM12->.) (Fig. 3). In the other 2 cases ( 8 5 K and 86K; correAbortive V^-gene rearrangements in A-expressing MM
sponding to MMI6-?. and MM13-^ respectively) of abortively
When studied by PCR performed on genomic DNA. 3/5 cases
rearranged V,, genes, a non-templated nucleotide was inserted
ot X LC-expressing MM tested were found to carry rearranged
in a site corresponding to amino acid position 37, rendering
V,-geiie sequence.s, 2 / 3 of these abortive V^ genes carried out-
the remaining V, seqttence out of frame. It is intriguing that in
of-frame rearrangements as a result of non-templated single
one ofthe abortively rearranged V,, genes studied by Rassenti et
base insertion in the FWR2 region at position 37 according to
al. (52), insertion of a non-templated nucleoiide occurred
Kabat's nomenclature (Fijj. 3), Tbe analysis of the third abor-
again in a site corresponding to amino acid position 37 in the
tively rearranged V.-gene sequence reveaied the presence of a
PA'R2. Therefore, this particular position ofthe V,- region may
stop codon in the FWR3 region at position 83 (Fig. 3), One case
represent a hotspot fbr mutations or extra base additions (7),
o(X LC-expressing MM was not analyzed for abortive V,-gene
The inabilit)' to amplify V, genes from genomic DNA in the
rearrangement due to the lack of genomic DNA material,
remaining two cases of X, LC-expressing MM in our smdy may
A high number of mutations was observed in the abor-
simply be due lo technical reasons or, alternatively, indicate
tively rearranged V, genes in 3/5 X LC-expressing MMs, How-
either that direct rearrangement of the V,, gene to Kde has
ever, tlieir distribution does in no way suggest selection by anti-
occurred inadvertently or that an "abortively" rearranged X:
gen. Taking into account the data of Rassenti et al. (52) and
gene has been deleted from the genome.
Wagner et al. (1 7) concerning somatic liypermutauon of nonfunctional (abortive) V., genes in X LC-expressing B-cell lymphoproliferative disorders, it becomes apparent that extensive
Conclusions
somatic mtitations occur exclusively in Waldenstrom's macro-
Transforming events in FL pathogenesis can occttr at different
giobulinemia (WM) and MM, Based botb on our findings and
stages of B-cell development within the GCs (5 5), It is therefore
the cited observations, the following conclusions can be
tempting to speculate that in certain FL subtypes the clontjgetiic
inferred: i) both functional and abortive V,, genes of post-
B cells' V,, genes have not yet been ihe target of the somatic
GC B cells are open to the somatic hypermutation machinery;
hypermutation machinery. In contrast, according to our data
ii} hyperiintiation ofthe V, genes tnigbt play an important role
(18. 56) and that of others (17. 19), the rearranged V, genes
in the antigen selection process operating at a B-cell stage
ofthe clonogenic B cells in MM always show extensive somatic
closely related to die progenitor of WM and MM, A recent anal-
mutations, with high R:S ratios in CDRs, implying that in this
ysis of out-of-frame V.,-gene rearrangements in X LC-expressing
malignancy the neoplastic B-cell precursor has already under-
HRS cells obtained from cases with HD revealed a characteristic
gone antigen selection and botl] its HC and LC have participated
paucity of tnutations in these non-ftmctional V,- genes (41),
to the process.
These findings, seen in the context of our data and that of Wag-
The differences in the mutational frequencies of the rear-
ner et al. (17), regarding abortive V,- genes, could strongly
ranged V^ genes between FL and MM may reflect differences in
imply that the differential accessibility of the V^-gene locus to
the ontogenetic B-cell siage at which neoplastic transformation Revims 162/1998
289
Kosmas et al • Hypermutation of V genes in FL and M M
had occurred. It appears that during the intra-GC life of a B cell the increased stringency requirements for selection by antigen are paralleled to a continuously increasing number of somatic mutations targeting Vn and VL genes. Flowever, a more delayed and limited pattern of somatic mutations within V^ genes might be a nseful marker in discriminating early versus late stages of B-cell ontogeny in the GCs. The finding of a considerably higher degree of VL-gene hypermutation, but without intraclonal diversity, in MM compared to FL raises some important questions concerning the postulated normal counterpart of the MM clonogenic B cell. As post-GC memory B cells are participating in the secondary immune response, proliferative expansion can take place without further somatic hypermutation followed by terminal differentiation into antibody-secreting plasma cells (57). This fact can provide a plausible explanation of the lack of ongoing somatic mutations within VH and VL genes in MM since its physiologic counterparts at this stage proliferate outside the GC, Certain memory B cells, however, may re-circulate tbrough the GC and undergo further rounds of somatic mutation and antigen selection (58—60), thus further improving and expanding B-cell memory. The latter could provide good evidence in order to construct a hypothesis regard-
ing accumulation of a higher number of somatic mutations within the V^ genes of clonogenic MM B cells. An important observation raised recently (i 9) pointed out that comparison of V,, and VL sequences derived from the same MM B-cell clone yielded significant clustering of mutations for antigen selection in CDRs of either the VH or X gene, but never in both. However, no physiological analog to this phenomenon has been observed at least when normal antigen-selected B cells are concerned. Similarly with regard to FLs in our analysis, 7/10 cases exhibited significant clustering ofmutations either in VH or V^ genes, but not in both, thus supporting the suggestion raised (19) that acomplementary imprint of antigen selection witnessed by V,, and VL tumor-derived sequences might constitute an important event during B-cell ontogeny It is believed tbat (he analysis of V-gene involvement in the process of antigen selection in variotis B-lymphopro!iferative disorders will shed light on the mechanisms underlying transformation and maintenance of the neoplastic clone; furthermore, it will hopefully help in defining the ontogenetic relationship between the neoplastic clonogenic B cell and its presumed normal B-cell counterpart.
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