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Blood, 15 September 2002, Vol. 100, No. 6, pp. 2269-2270
CORRESPONDENCE
To the editor:
Glycosylation of V region genes in follicular lymphoma
as a result of the somatic hypermutation mechanism
We read with interest the report by Zhu et al,1 who
examined the generation of potential N-glycosylation sites in the
clonogenic immunoglobulin (Ig) heavy chain (VH) genes of
patients with follicular lymphoma (FL).1 Using their own
and a number of sequences derived from the published literature, these
authors have investigated the frequency of potential N-glycosylation
sites introduced into functional VH genes as a consequence
of somatic mutation. FL cells were compared with normal memory B cells
or plasma cells matched for similar levels of mutation. Strikingly,
novel sites were detected in almost 80% of patients with FL, compared
with 9% in the normal B-cell population (P < .001),
whereas diffuse large B-cell lymphoma (DLCL) showed an intermediate
frequency (41%). Multiple myeloma (MM) and the mutated subset of
B-cell chronic lymphocytic leukemia (B-CLL) showed frequencies similar
to those of normal cells in 8% and 13% of patients, respectively. The
authors concluded that N-glycosylation of the VH region may
be a common event in FL and in a subset of DLCL, with the novel sites
predominantly located in complementarity-determining regions
(CDRs), the major target of the somatic hypermutation machinery.
Sequences of nonfunctional VH genes contained few sites,
arguing for positive selection for novel glycosylation sites in
functional FL VH sequences. Moreover, the authors suggested
that the added carbohydrate in the VH region might
contribute to interaction with antigenic elements in the germinal
center (GC) environment, and that this common feature of FL may be
critical for tumor behavior. We conducted a similar analysis for novel glycosylation sites in our
published series of VH and V sequences from
FL2 and MM patients.3 Novel glycosylation
sites were identified in 4 out of 10 FL VH genes; there
were 2 novel sites in the CDR2 and one each in the FWR1 and FWR2
regions. In MM V and V genes, novel
glycosylation sites were detected in only 2 out of 17 cases (11.7%);
both sites were located in V CDR1 region. Our findings
are generally in accordance with those by Zhu et al1
regarding MM. However, markedly different results were obtained from
analysis of FL V genes; Zhu et al reported an overall
incidence of almost 59% (10/17 cases; Table 1 of their article),
whereas no novel glycosylation sites were identified in our published sequences. FL can be considered as the neoplastic counterpart of a certain stage
in the intra-germinal center maturation of B cells.4 At
this stage, normal B cells that are specific for antigen (with surface
Ig [sIg] exhibiting an affinity above the germ line potential) are
subject to positive selection.5 Sequence analysis of
rearranged VH and V genes of FL cells
carried out by our group demonstrated that whereas rearranged
VH genes were hypermutated in their majority, with
clustering of mutations in the CDRs, the rearranged V genes of the same neoplastic clones varied significantly with regard to
their mutational profile; indeed, very few or no mutations were
observed in a significant number of cases (5/10; 50%) of the analyzed
V sequences.3 A similar pattern is obtained from the study by Zhu et al;1 although the
V genes of that study were, on average, more heavily
mutated than the corresponding genes in our study, the median
"mutation load" in V sequences was still
significantly smaller than in the same cohort's VH genes (median homology to germ line was 89% for VH genes versus
96% for V genes). The fact that clonogenic
V genes are less frequently mutated indicates that the
somatic hypermutation machinery might have ceased to operate in the
V locus at the time when neoplastic transformation had
occurred. Perhaps this is a reflection of the generally smaller
potential contribution of V genes (compared with
VH genes) in antigen selection of the clonogenic B cells in
FL.3 Similar conclusions regarding the role of Ig light
chains in antigen recognition (evidenced by the mutation status of the
corresponding V region genes) have been reached from single-cell
studies in the normal peripheral B-cell repertoire, indicating a more
limited mutational load both in the expressed as well as nonfunctional
V genes compared with their partner VH genes
in IgM+/CD5 B cells.6 In conclusion, the study of Zhu et al,1 as well as
analysis of our previously published series, refocuses the interest of
antigen selection in human lymphomagenesis to the potential contribution of Ig V region glycosylation at novel sites as a result of
active somatic hypermutation. However, until these results are accepted
as definite, further confirmatory studies in larger numbers of patients
are awaited.
Chrysoula Belessi, Kostas Stamatopoulos, and Christos Kosmas
Correspondence: Christos Kosmas, Metaxa Cancer Hospital,
Medicine-2nd Division of Medical Oncology, 21 Apolloniou St, 16341 Athens, Greece; e-mail: ckosm{at}ath.forthnet.gr
References
1.
Zhu D, McCarthy H, Ottensmeier CH, Johnson P, Hamblin TJ, Stevenson FK.
Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma.
Blood.
2002;99:2562-2568[Abstract/Free Full Text].
2.
Stamatopoulos K, Kosmas C, Papadaki T, et al.
Follicular lymphoma immunoglobulin  light chains are affected by the antigen selection process, but to a lesser degree than their partner heavy chains.
Br J Haematol.
1997;96:132-146[CrossRef][Medline]
[Order article via Infotrieve].
3.
Stamatopoulos K, Kosmas C, Belessi C, Stavroyianni N, Kyriazopoulos P, Papadaki T.
Molecular insights into the immunopathogenesis of follicular lymphoma.
Immunol Today.
2000;21:298-305[CrossRef][Medline]
[Order article via Infotrieve].
4.
Kosmas C, Stamatopoulos K, Papadaki T, et al.
Somatic hypermutation of immunoglobulin genes: focus on follicular lymphoma and multiple myeloma.
Immunol Rev.
1998;162:281-292[CrossRef][Medline]
[Order article via Infotrieve].
5.
Diaz M, Casali P.
Somatic immunoglobulin hypermutation.
Curr Opin Immunol.
2002;14:235-240[CrossRef][Medline]
[Order article via Infotrieve].
6.
Foster SJ, Brezinschek HP, Brezinschek RI, Lipsky PE.
Molecular mechanisms and selective influences that shape the kappa gene repertoire of IgM+ B cells.
J Clin Invest.
1997;99:1614-1627[Medline]
[Order article via Infotrieve].
Response:
High incidence of novel N-glycosylation sites in the
immunoglobulin variable region genes of follicular lymphoma
Our finding of a high frequency of potential glycosylation sites
in the variable regions of cases of follicular lymphoma (FL) has
implications for the understanding of the pathogenesis of this
tumor.1 The presence of oligosaccharides in the binding site of the B-cell receptor of FL cells may influence interactions with
stromal elements in the germinal center and could contribute to tumor
growth or survival. Clearly the data on 70 cases in our paper need to
be extended, and Belessi et al also make this point. The sites are
largely acquired by somatic mutation and can be found in either
VH or VL. One problem in extending the data is that generally only VH sequences are available from the
databases, leaving open the question of the contribution of sites in
VL. Our paper included matched VH and
VL sequences from 17 cases from this laboratory, and we
have now added a further 11 matched cases, all of which had sites. In
summary, we now have 26 of 28 cases with sites in VH and 14 of 28 with sites in VL, and all our cases had at least one
site. An interesting exception was a case initially diagnosed as FL,
but with no sites evident. We asked a pathologist (Professor Dennis
Wright) to give a second opinion, and he made the diagnosis of mantle
cell lymphoma, subsequently confirmed by staining for cyclin D1. While
this does not yet allow us to conclude that all cases of FL will have
sites, it does indicate that diagnostic criteria must be met.
Disturbingly, Belessi et al have added 10 cases, and only 4 of these
have sites, all of which were in VH. While this is a small
number, it does not fit with our observations, and we await further
findings from our cases and from the community. Belessi et al then focus on events occurring in VK, where
they have sequence data on 10 cases. They make the point that levels of
somatic mutation are lower in VL than VH, an
observation that we confirm in our set of sequences, which had 89% and
96% homology for VH and VK,
respectively.1 This discrepancy has been reported in
normal B cells and in other B-cell malignancies, eg, in Burkitt lymphoma.2 In relation to acquisition of glycosylation
sites, it is obvious that they will be less likely to accumulate in
VL, and, in fact, 5 of 10 of the cases analyzed by Belessi
et al were close to germ line sequence. However, a low mutational rate
may be sufficient since, in one of our cases with 99% homology in VK, a site had been acquired.1 Belessi et al
speculate on the reason for the differential mutational rate in
VH and VL, and they suggest that "the somatic
hypermutation machinery might have ceased to operate in the
VK locus at the time when neoplastic transformation had
occurred." In our view this is unlikely, given that intraclonal
variation continues to accumulate in VK sequences in FL
(Zhu et al3 and our unpublished observations, May
2002). An alternative explanation for the discrepancy in
mutational level may be a lower frequency of sequence motifs that
surround mutational hot spots,4 or a difference in the
elements outside the coding region that influence somatic
mutation.5 The role of germ line VL sequence
in antigen recognition has been assumed to be less than that of
VH, due to the lack of D-segment genes. The relatively low
level of somatic mutation would support this lesser role. However,
crystallographic analysis clearly implicates VL in antigen recognition,6 and the degree of involvement is likely to
vary for different antibodies. Generalizations about the immensely variable structures of the binding site of antibodies may be difficult, but the presence of oligosaccharides, largely avoided by normal B
cells, could be telling us something about B-cell tumors. Since there
may be consequences for new treatment options, we await data from more
matched sequences of FL with considerable interest.
Delin Zhu, Helen McCarthy, Christian H. Ottensmeier, Peter Johnson, Terry J. Hamblin, and Freda K. Stevenson
Correspondence: Delin Zhu, Molecular Immunology Group,
Tenovus Laboratory, Cancer Sciences Division, Southampton University
Hospitals Trust, Southampton SO16 6YD, United Kingdom.
References
1.
Zhu D, McCarthy H, Ottensmeier CH, Johnson P, Hamblin TJ, Stevenson FK.
Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma.
Blood.
2002;99:2562-2568[Abstract/Free Full Text].
2.
Chapman CJ, Wright D, Stevenson FK.
Insight into Burkitt's lymphoma from immunoglobulin variable region gene analysis.
Leuk Lymphoma.
1998;30:257-267[Medline]
[Order article via Infotrieve].
3.
Zhu D, Hawkins RE, Hamblin TJ, Stevenson FK.
Clonal history of a human follicular lymphoma as revealed in the immunoglobulin variable region genes.
Br J Haematol.
1994;86:505-512[Medline]
[Order article via Infotrieve].
4.
Betz AG, Neuberger MS, Milstein C.
Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes.
Immunol Today.
1993;14:405-411[CrossRef][Medline]
[Order article via Infotrieve].
5.
Klix N, Jolly CJ, Davies SL, Bruggemann M, Williams GT, Neuberger MS.
Multiple sequences from downstream of the J kappa cluster can combine to recruit somatic hypermutation to a heterologous, upstream mutation domain.
Eur J Immunol.
1998;28:317-326[CrossRef][Medline]
[Order article via Infotrieve].
6.
Tello D, Goldbaum FA, Mariuzza RA, Ysern X, Schwarz FP, Poljak RJ.
Three-dimensional structure and thermodynamics of antigen binding by anti-lysozyme antibodies.
Biochem Soc Trans.
1993;21:943-946[Medline]
[Order article via Infotrieve].
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