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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2922-2929
NEOPLASIA
From the Department of Pathology, Academic Medical Center,
Amsterdam; the Department of Pathology, Radboud Hospital, Nijmegen; the
Department of Pathology, Leiden University Medical Center, Leiden; and
the Department of Pathology, the Westeinde Hospital, The Hague, the
Netherlands.
The expansion of follicular lymphomas (FLs) resembles, both
morphologically and functionally, normal germinal center B-cell growth.
The tumor cells proliferate in networks of follicular dendritic cells
and are believed to be capable of somatic hypermutation and isotype
switching. To investigate the relation between somatic mutation and
heavy chain isotype expression, we analyzed the variable heavy
(VH) chain genes of 30 FL samples of different
isotypes. The VH genes of the FLs were heavily mutated
(29.3 mutations on average). In addition, isotype-switched lymphomas
contained more somatic mutations than immunoglobulin M-positive
lymphomas (33.8 mutations per VH gene versus 23.0, respectively). In all but one of the FLs, the ratios of replacement
versus silent mutations in the framework regions were low, independent
of the absolute number of somatic mutations and the level of
intraclonal variation. Analysis of relapse samples of 4 FLs showed no
obvious increase in somatic mutation load in most FLs and a decrease in
intraclonal variation in time. In 3 of 4 cases, we obtained evidence
for selection of certain subclones, rather than clonal evolution. Our
findings question if intraclonal variation is always a reflection of
ongoing somatic hypermutation. This may have implications for the
concept of antigen-driven lymphomagenesis.
(Blood. 2000;95:2922-2929)
Follicular lymphoma (FL) is one of the most common
B-cell non-Hodgkin lymphomas in adults in Europe and the United
States.1 FLs have a relatively indolent behavior, with a
reported median survival of 8 to 10 years after
diagnosis.2-4 At the molecular level, FLs are characterized
by the t(14;18)(q32;q21) translocation, which can be demonstrated in
approximately 80% to 90% of FLs.5,6 Due to this
translocation, the BCL-2 protein becomes constitutively expressed,
which protects the cells against the induction of apoptosis and is thus
believed to be of pathogenetic relevance.7,8 On the other
hand, the t(14;18) translocation alone is not sufficient for full
transformation, as has been demonstrated in bcl-2 transgenic mice.9 Moreover, the t(14;18) translocation is also found
in normal B cells of healthy individuals.10-12 Additional
genetic aberrations must therefore be present and have in fact
been described.13,14
FLs are histologically well differentiated. Their nodular growth
pattern clearly resembles the architecture of germinal centers in
secondary lymphoid organs.15 The neoplastic follicles
contain centroblastic and centrocytic tumor cells as well as
nonneoplastic T cells, follicular dendritic cells, and few, if any,
macrophages.16,17 Analyses of the variable heavy
(VH) and light (VL) chain genes of the B-cell
antigen receptor (BCR) of FLs have further supported their derivation
from germinal center B cells: The immunoglobulin (Ig) V genes of FLs
are somatically mutated,18,19 and the mutation patterns
observed are reminiscent of those of normal, antigen-selected, memory B
cells. In addition, intraclonal diversity in the mutated IgV genes of
individual FLs is well documented 20-23 and is generally considered to be a reflection of ongoing somatic hypermutation. Finally, heavy (H) chain isotype switching has been described for 2 cases of FL,24,25 an event that is thought to take place normally in the centrocyte stage of the germinal center.26
Thus far, studies have indicated that the IgV genes of FLs contain high
somatic mutation loads: Bahler et al reported an average frequency of
mutation of 8.6% (amounting to approximately 25 mutations) in
VH4-expressing FLs.18 This is in accordance
with a study by Stamatopoulos et al in which an average mutation
frequency of 9.3% (approximately 27 mutations) was observed. Also, the
In this study, we investigated the relation between the amount and
patterns of somatic mutations and the expressed H chain isotypes by
analyzing the BCRs of 12 IgM+(IgD+), 16 IgG+, and 1 IgA+ FL samples. One of these
lymphomas contained both IgM+ and IgG+ tumor
cells. Moreover, we investigated the changes over time in the
VH genes of 4 patients who suffered a relapse.
Patient material
Immunohistochemistry
RNA isolation and cDNA synthesis Total cellular RNA was isolated from frozen tissue sections using the Trizol reagent (Life Technologies, Breda, the Netherlands). For complementary DNA (cDNA) synthesis, 10 µg of RNA was incubated with 5 nmol of pd(N)6 primer (Pharmacia Biotech, Roosendaal, the Netherlands) for 10 minutes at 65°C. After cooling on ice, the reaction mixture was added to a final volume of 50 µL. It contained 400 units of Moloney murine leukemia virus reverse transcriptase (RT) (Life Technologies), 8 mM of dithiothreitol, 1 mM of each dNTP, 1 × first strand buffer (50 mM of Tris-HCl, pH 8.3; 75 mM of KCl; 3 mM of MgCl2), and 60 units of RNAse inhibitor (Boehringer Mannheim, Almere, the Netherlands). The reaction was performed for 1 hour at 37°C. Subsequently, the enzyme was inactivated during 10 minutes at 95°C.Polymerase chain reactions The complementarity-determining region 3 (CDR3) was amplified using a forward primer with specificity for framework region 3 (FR3) in combination with reverse primers specific for JH (JHseq), Cµ, C (C2), C , or C , as
described.27 Either 1 µL of the cDNA reaction mixture was
used, or (for a nested polymerase chain reaction [PCR]) 1 µL of PCR
product from a VH family-specific PCR was used. The PCR
mixture contained 1 × Taq buffer (20 mM of Tris-HCl, 50 mM of
KCl, pH 8.4), 0.2 mM of each dNTP, 1.5 mM of MgCl2, 2 units
of Taq polymerase (Life Technologies), and 0.5 µM of each primer.
First, 10 cycles of amplification were performed in the thermal cycler
(PTC-100, MJ Research Inc, Watertown, MA), ie, successively 30 seconds
at 95°C, 20 seconds at 57°C, and 20 seconds at 72°C. The
next 40 cycles of amplification consisted of 30 seconds at 95°C, 20 seconds at 55°C, and 20 seconds at 72°C. The reaction was
completed for 6 minutes at 72°C. PCR products were analyzed on a
3% Metaphor agarose gel (FMC Bioproducts, Rockland, ME). For the
VH family-specific PCR, reactions were performed with one
of the VH family-specific leader primers combined with the
appropriate reverse primer, either JH, Cµ, C, or
C.27 The PCR reaction mixture was the same as for the
CDR3-specific PCR except that 1 unit of Taq polymerase and 0.25 µM of
each primer was used. Thirty cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C were performed. The
reaction was terminated for 6 minutes at 72°C. The PCR products
were analyzed on a 1% standard agarose gel (Sigma, St. Louis, MO).
Cloning and sequencing of PCR products PCR products were cloned into pGEM-T vectors (Promega, Leiden, the Netherlands) and transformed into DH10b bacteria (Life Technologies). Subsequently, from 4 or more colonies both strands of the inserts were sequenced to obtain the sequence of the dominant clone, the consensus sequence. Sequencing was performed with an ABI sequencer (Perkin Elmer Corp, Norwalk, CT) using the dye-terminator cycle-sequencing kit (Perkin Elmer). To determine the Taq error rate of our experimental design, 19 clones of CD79a and CD79b were sequenced. These clones were generated according to the same PCR and cloning procedures as used for the VH genes. The Taq error frequency thus established is 0.14%, which amounts to 0.4 mutation/VH clone (data not shown).Assignment of mutations The sequences found were compared with published germline sequences using the Vbase database28 and DNAplot29 on the Internet (http://www.mrc-cpe.cam.ac.uk/imt-doc) to identify mutations. The last nucleotide position of the V gene was excluded from the mutational analysis in view of possible nucleotide deletions at the joining sites.Statistical analysis To calculate whether the excess or scarcity of replacement (R) mutations in the FRs had occurred by chance, we used the binomial distribution model as proposed by Chang and Casali.30 If the B cell was selected for antibody expression, there is a counterselection for R mutations in the FRs. The ratios of R versus silent (S) mutations (R/S) found in the CDRs are often higher than expected but are not used by us as arguments for or against antigenic selection; Dörner et al showed that the R/S values of both FRs and CDRs were higher in nonproductive and, therefore, not antigen-selected rearrangements than in productive, antigen-selected rearrangements.31 Also, additional R mutations in the CDRs can be unfavorable in already selected immunoglobulin with high affinity.32 To calculate the significance of the differences in the amounts of somatic mutation in IgM+ and isotype-switched FLs, the Mann-Whitney 2-sample test was used. In this analysis, every tumor sample was included as a separate entity.
Clonality assessment and isotype expression Of the selected panel of FLs, clonality and isotype expression were established immunohistochemically. To confirm this at the molecular level, the CDR3 region was amplified with a FR3 primer in combination with primers specific for the JH gene or the 5' regions of Cµ, C (C2), C, or C.27
With this PCR we can assess clonality and isotype expression with a
sensitivity of about 25% clonal cells in a polyclonal background. In
30 of 59 FLs, we observed a sharp band on agarose gel with both the JH primer and 1 or 2 of the primers specific for the
constant regions as downstream primers. In another 13 cases, we could
establish clonality only with a Cµ, C, C, or C downstream
primer and not with the JH primer. In summary, in 43 FLs
(72.9%) we could confirm clonality with a CDR3-specific PCR. This
percentage is similar to what has been reported by others for PCR
clonality assays of FLs.33-35 Of these cases, we chose 30 FLs for our analyses of the VH gene sequences (Table
1). In this selection, isotype-switched FLs
were relatively overrepresented. In 3 cases, immunohistochemical determination of the expressed H chain was not clear (nos. 6, 25, and
56), whereas by RT-PCR clonal bands with the C2-specific primer were
obtained (not shown). In summary, 12 FLs expressed IgM, of which at
least 10 coexpressed IgD at the RNA level; 13 FLs expressed IgG; and 1 FL expressed IgA (Table 1). Interestingly, in 1 of these lymphomas, no.
8-'83, both IgM- and IgG-expressing tumor cells were found by
immunofluorescence (not shown). In accordance, CDR3-specific RT-PCR
yielded sharp bands in the lanes corresponding to the JH,
Cµ, C, and C downstream primers (Figure
1). Sequencing of the VH-Cµ
and the VH-C PCR products showed that both tumor cell
populations contained the same VDJ rearrangement (Table 1). A relapse
sample of this patient contained only the IgG-expressing tumor cell
population, assessed immunohistochemically and by CDR3-specific PCR
(Figure 1).
VH, D, and JH gene usage The complete VH genes were amplified with family-specific VH leader primers in combination with a JH primer or constant H chain isotype-specific primers. To ascertain that these VH products originated from the tumor population, we performed a nested CDR3-specific PCR on the VH products obtained. In all cases, the nested CDR3 products and the original CDR3 products had identical sizes. Next, the VH products were cloned and sequenced. The number of somatic mutations was determined by comparing the VH sequences to the germline genes with the highest homology (Table 1). Fifteen FLs used genes from the VH3 family (60%). VH4 and VH1 family genes were found in 6 FLs (24%) and 2 FLs (8%), respectively. VH5 and VH7 genes were each found once (4%). The JH4b gene was found in 9 FLs, JH6 genes were found in 6 FLs, and JH3 and JH5 genes were each used by 4 FLs. The JH1 and JH2 genes were each found once. A stretch of 7 consecutive nucleotide matches was taken as an indication that a certain D gene was used. According to this method, only 6 D genes could be assigned (Table 2).
Number of somatic mutations Most FLs, including the IgM-expressing FLs, were heavily mutated (Table 1). Still, in this selection of FLs we observed a statistically significant difference in the number of somatic mutations in IgM-expressing versus isotype-switched FL samples (P < .016; Figure 2). On average, FL samples of IgM isotype contained 23.0 somatic mutations (7.8%), ranging from 8 to 49 somatic mutations. The FL samples of IgG isotype contained an average of 34.1 somatic mutations (11.6%), with a range from 8 to 51 somatic mutations. The IgA-expressing FL harbored 29 somatic mutations (9.9%; Table 1). These differences in mutation load are also obvious from calculations on individual molecular clones of these lymphomas: When for each clone the amount of somatic mutations compared with the germline gene was calculated and when these amounts were averaged between the clones, the difference in somatic mutation loads amounted to 34.6 mutations per VH gene for isotype-switched FLs and 23.6 mutations for IgM+ FLs (data not shown). In these clonal analyses, however, Taq errors are included ( 0.4 mutations per clone).
Intraclonal variation To assess the degree of intraclonal variation, independent clones of 21 FLs were sequenced (Table 1). In 15 of these FLs, intraclonal variation above the Taq error frequency was observed, ranging from 0.5 to 14.6 mutations per clone. Moreover, in 11 of these 15 lymphomas, mutations were found that were shared by more than 1 clone (data not shown), which argues strongly for somatic hypermutation rather than Taq error. No obvious difference in the level of intraclonal variation in IgM- and IgG-expressing FLs was observed. In 6 FLs (nos. 3-'93, 3-'95, 15, 25, 17, and 57) the intraclonal variation did not exceed the Taq error rate of 0.14%, which amounts to 0.4 mutations per clone.Mutation patterns To assess whether the FLs with variable levels of mutation and
intraclonal diversityshowed signs of selection for a potentially functional BCR, the distribution of R and S mutations in FRs and CDRs
was analyzed. For this purpose, the dominant or "consensus" VH sequences of individual FLs were analyzed. The ratio of
R/S mutations was highly variable in the CDR1 and CDR2 (average 3.9), whereas in the FRs the R/S ratio was consistently lower than 2.0 (average 0.97), with 1 exception (FL no. 59; Table
3 and Figure 3). When the distribution of R and S
mutations was analyzed according to the binomial distribution model by
Chang and Casali,30 the number of R mutations in the FRs of
most FL samples was significantly lower than would be expected if the
mutations had occurred at random and in the absence of selective
forces. The nature of the R mutations (conservative or nonconservative)
was not different between the CDRs and FRs. These data clearly indicate
that in FLs the overall structure of the BCR is maintained irrespective of the total number of somatic mutations and the degree of intraclonal variation (Table 3 and Figure 3). Only in lymphoma no. 59 was the R/S
value in the FR exceptionally high: 9 R mutations versus 1 S mutation
were found in the FRs, 5 of which occurred in the FR1. The absolute
number of R mutations in the FRs, however, did not differ much from
some other FLs. Furthermore, because expression of surface
IgG was found immunohistochemically (Table 1), the high amount of R
mutations in the FRs apparently did not interfere with BCR expression.
Isotype switch variants and relapse of follicular lymphoma no. 8 In FL no. 8-'83, both IgM- and IgG-expressing tumor cells were found, which harbored the same VDJ rearrangement. Compared with the germline gene V3-23, the IgM-related sequence contained 30 somatic mutations, whereas the IgG-related sequence contained 35 somatic mutations (Table 1, Figure 4). The patient was treated with chemotherapy and achieved complete remission. A local relapse 5 years later was irradiated, and 9 years after the first presentation a systemic relapse developed (no. 8-'92), which was again treated chemotherapeutically. This relapse consisted of IgG-expressing tumor cells only. Compared with the germline gene, also 35 somatic mutations were found, of which 30 somatic mutations were shared with the IgM- and IgG-related sequence of the first time point, and 5 were different (Figure 4). In all VH gene sequences of lymphoma no. 8, a deletion of codon 3 was observed (not shown), which is most likely due to somatic mutation.36,37
Relapses of follicular lymphomas no. 3, 6, and 66 Patient no. 3, who initially achieved complete remission after chemotherapy, developed a relapse after 2 years. In the relapse sample, the total amount of somatic mutations in the VH gene was lower than in the presentation biopsy, 21 and 26 mutations, respectively. Compared with the germline gene V3-7, 19 mutations were found that were shared between the 2 time points (Table 1 and Figure 4). No significant intraclonal variation was found at either time point. Patient no. 6 presented with a stage IV, low-grade FL. No treatment was given ("watchful waiting"). After 4 years, a second lymph node was resected because of clinical progression. The total number of somatic mutations compared with the germline gene V3-23 was 44 in the presentation biopsy, compared with 50 somatic mutations in the second sample (Table 1 and Figure 4). In the samples of both time points, intraclonal variation above the Taq error was found, but in time the intraclonal variation had decreased from 2.8 to 1.8 mutations per clone. Patient no. 66 developed 2 relapses, 1 and 2 years after the presentation of the FL in 1996, respectively. Chemotherapeutic treatment started in 1997 after clinical progression, upon which only partial remission was achieved. The somatic mutation loads in the samples of these 3 time points were 38, 39, and 40 mutations, respectively, whereas the intraclonal variation gradually decreased from 3 mutations per clone to 0.5 mutations per clone. Curiously, although the absolute amount of somatic mutations increased, the location of the mutations was not the same in samples of these 3 time points (Figure 4). Compared with the germline gene, 37 somatic mutations were shared between the 3 time points. In the presentation biopsy, 1 additional R mutation was found at codon 23. This mutation was not found in the sample of the second time point. In addition, 2 other R mutations were found at codon 57 and 64. In FL no. 66-'98, except for the 37 shared mutations, a new R mutation was found at codon 5. The mutation found at codon 23 was shared with the first time point, whereas the mutation at codon 64 was shared with the second time point (Figure 4). Also, at the clonal level only a slight increase was found in the number of somatic mutations: The average number of somatic mutations of the clones compared with the germline gene V4-39 was 39.7 in the samples of the first 2 time points and 40.5 in 66-'98 (data not shown).
In this study, the VH genes of 30 FL samples of different isotypes were analyzed. In agreement with Bahler et al,18 the VH family gene usage of the FLs studied was not obviously different from VH gene usage reported for normal B cells.38,39 Compared with normal postfollicular B cells derived from tonsil, spleen, or lymph node, the VH genes of FLs contained significantly more mutations, which is compatible with prolonged expansion in a germinal center-like environment. We observed mutation numbers ranging from 8 to 51 (2.7% and 17.2%, respectively), with an average of 29.3 (9.9%) per VH gene. Similar frequencies have been reported previously by others.18,19 In 15 of the 21 FLs studied at the clonal level, intraclonal variation above the Taq error frequency was found, varying from 0.5 to 14.8 mutations per cloned VH gene (Table 1).
The authors thank N.J. Ponne for technical assistance with sequencing, J.B.G. Mulder for assistance with immunohistochemistry, and J.W. Vaandrager and C.C.H. Vellema for kindly collecting and providing tissue material from FLs. M. Ek and Dr C.E.M. Hollak are thanked for providing clinical data. We also thank Dr R. Küppers for providing intrinsic R/S values of several VH genes, Dr A.C. Tersmette for advice on statistics, and Dr O.J. de Boer for designing a computer program to calculate the probability of the R/S distributions found.
Submitted June 23, 1999; accepted January 6, 2000.
Supported by a grant from the Dutch Cancer Society (Grant no. AMC 95-957). C.J.M.v.N. is a fellow of the Netherlands Royal Academy of Arts and Sciences.
Reprints: C. J. M. van Noesel, Department of Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, the Netherlands; e-mail: c.j.vannoesel{at}amc.uva.nl.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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W. M. Aarts, R. J. Bende, J. G. Bossenbroek, S. T. Pals, and C. J. M. van Noesel Variable heavy-chain gene analysis of follicular lymphomas: subclone selection rather than clonal evolution over time Blood, July 1, 2001; 98(1): 238 - 240. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
light chains used by these lymphomas were mutated, although to a
lesser extent: 4.7% mutation was found on average (amounting to
approximately 13 mutations per VL gene).
indicates the R/S value of the
CDRs; 
indicates the R/S value of the FRs.
represents an R mutation. The codon numbers in which mutations took
place are indicated under these symbols. The total number of somatic
mutations for each FL sample (in bold) and the intraclonal variation,
in number of mutations per clone, is indicated next to each line.