Leukemia (2001) 15, 1772–1778  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Somatic hypermutation targeting to intrinsic hotspots of immunoglobulin genes in follicular lymphoma and multiple myeloma C Belessi1, K Stamatopoulos2, N Stavroyianni2, K Zoi2, T Papadaki3 and C Kosmas4

1Hematology Laboratory, General Hospital of Nikea, Piraeus; 2First Department of Medicine, Athens University School of Medicine, Laikon General Hospital, Athens; 3Hemopathology Unit, Evangelismos Hospital, Athens; and 4Department of Medicine, Helena Venizelou Hospital, Athens, Greece

In this study, we analyzed the targeting of the somatic hyper- of the secondary lymphoid organs where contact with and mutation (SHM) mechanism at specific hotspot sequence 3 ␬ selection by antigen takes place. The molecular hallmark of motifs in the VH and V genes of 10 follicular lymphoma (FL) cases and the V␬ and V␭ genes of 11 ␬- and six ␭-light chain this phase is the introduction of mutations within rearranged expressing multiple myeloma (MM) cases. These sequences IgV genes, at a rate much higher than usual, a phenomenon were analyzed for targeting of specific motifs, ie certain highly described as somatic hypermutation.4 mutable trinucleotides (3-NTPs), the tetranucleotide (4-NTP) Antigen selection in B cell ontogeny is evidenced by non- RGYW and its complementary, WRCY (where R = purine, random distribution of somatic mutations in lg HC and LC V = = Y pyrimidine and W A or T). Comparisons were carried out genes, a feature providing useful information concerning the between mutation frequencies in RGYW vs WRCY and the inci- 5 dence of mutations in complementarity determining region ontogenetic assignment of B cell neoplastic disorders. In this (CDR)-1 vs CDR2 vs CDR3. Statistically significant differences context, an increased ratio of replacement (R) to silent (S) were obtained when comparing: (1) the ratio of mutations in 4- mutations in the complementarity determining regions (CDRs) NTPs (RGYW, WRCY, RGYW+WRCY)/mutations in the whole V of IgV genes, has been considered as the most reliable surro- sequence in MM-V␬ vs MM-V␭; (2) the total number of mutated gate marker of selection by antigen for higher avidity.6 How- ␬ ␬ 4-NTPs in MM-V vs FL-V ; (3) the number of mutated RGYW ever, irrespective of subsequent selection, somatic hypermut- 4-NTPs in MM-V␬ vs FL-V␬ and FL-VH vs FL-V␬; (4) the number of mutated WRCY 4-NTPs in MM-V␬ vs FL-V␬ (P = 0.006) and ation is primarily targeted at specific hotspots within V genes, FL-V vs FL-V␬; (5) the targeting of RGYW vs WRCY in the ie tri-or tetra-nucleotidesequence motifs, such as the RGYW H = = = CDRs of FL-VH genes. Similar results (regarding statistical motif (R purine, Y pyrimidine, W A or T) and its significance) were obtained when undertaking intergroup com- complementary, WRCY.7 parisons for 3-NTPs. These findings conform well with relevant While the exact mechanism of somatic hypermutation data derived from normal peripheral B cells. The differences remains elusive, this phenomenon is characterized by certain observed in favor of 4-NTP (RGYW and WRCY) targeting in FL- 8 ␬ ␬ ␬ unique features. The nature of mutations indicates a prefer- VH vs FL-V and MM-V vs FL-V may implicate differences in the evolution of SHM coupled with selection in different stages ence for transitions over transversions with purines being of B cell ontogeny. Several explanations can be offered for the more frequently targeted than pyrimidines, suggesting strand fact that hotspot sequences were not always targeted by SHM bias; mutations are concentrated mainly in the CDRs and most in FL and MM: (1) other unrecognized motifs may be targets of often are single nucleotide substitutions rather than deletions SHM; (2) ‘inappropriately’ introduced mutations were fixed and or insertions; certain codons are targeted more often by the propagated by the neoplastic process; (3) certain FL and MM cases might have lost their ability to correct mutations intro- mutational process, while others are less likely to tolerate duced in classic hotspots due to deficient mismatch-repair changes; finally, a striking bias exists for G and C over A and (MMR) mechanisms; conversely, in other cases with intact T nucleotide mutations.9 MMR function, the hotspot to non-hotspot targeting of somatic In the present study, we analyzed the distribution of somatic hypermutation is balanced. Leukemia (2001) 15, 1772–1778. hypermutation and its targeting at specific mutational hotspots Keywords: immunoglobulin genes; hypermutation; follicular lym- in the clonotypic V and V␬ genes of follicular lymphoma (FL) phoma; multiple myeloma H as well as the V␬ and V␭ genes of multiple myeloma (MM), tumors corresponding to antigen-selected intra-germinal center (GC) and post-GC stages of B cell ontogeny. Introduction

The antigen-independent phase of B cell ontogeny takes place Materials and methods in the bone marrow and is characterized by ordered immuno- globulin (lg) gene rearrangements leading to the assembly of Sequence analysis distinct variable (V), diversity (D) (for heavy chains only) and joining (J) gene segments into a V(D)J gene complex, a process Included in the present study were the clonotypic V and V␬ 1 H known as V(D)J recombination. Successful rearrangement of gene sequences of 10 FL cases10 as well as the clonotypic V␬ heavy chains (HC) lg genes and subsequently that of light and V␭ gene sequences of 11␬-and six ␭-light chain express- ␬ ␭ chain (LC) lg genes ( or ) will enable the developing B cell ing MM cases analyzed previously by our group.11,12 The to later express on its surface a fully functional lg receptor sequences have been submitted to the EMBL database 2 with unmutated V region sequences. (http://www.ebi.ac.uk/embl/index.html) with the following The second, antigen-dependent, phase will start when the accession numbers: for FL-VH, AJ410896 to AJ410905; for FL- ‘naı¨ve’ B cell exiting the bone marrow enters into the follicles V␬, AJ410886 to AJ410895; for MM-V␬ AJ410906 to AJ410916; and for MM-V␭, AJ410917 to AJ410922. The analysis aimed at determining whether specific nucleo- Correspondence: C Kosmas, Department of Medicine-Oncology Unit, Helena-Venizelou Hospital, 21 Apolloniou Street, 163 41 Athens, tide motifs, ie the tetranucleotide RGYW and its complemen- Greece; Fax: 30.1.9962917 tary WRCY were targeted by the somatic hypermutation Received 31 January 2001; accepted 28 June 2001 machinery. A further objective was to identify whether the Hypermutation in Ig V genes of FL and MM C Belessi et al 1773 Table 1 Numbers of mutations in and mutated tetra-and tri-nucleotidesand their distributions in each CDR, FWR (framework region), entire V sequence, and RGYW/WRCY motifs

FWR1 FWR2 FWR3 CDR1 CDR2 CDR3 FWRS CDRS Total

Mutations/4NTPS FL-kappa 1/4 4/12 9/38 15/28 2/20 10/27 14/54 27/75 41/129 FL-heavy 9/43 7/29 23/83 17/25 50/101 39/155 67/126 106/281 MM-kappa 0/9 17/30 29/109 36/57 9/34 20/48 46/148 65/139 111/287 MM-lambda 1/9 10/32 13/76 16/35 5/23 11/40 27/117 32/98 60/215

4NTPS/Mutated FL-kappa 1/10 4/63 8/66 13/42 2/11 10/28 13/139 25/81 38/220 FL-heavy 10/56 5/22 24/53 16/28 36/52 39/131 52/80 91/211 MM-kappa 0/11 21/72 24/72 33/53 6/17 12/24 45/155 51/94 96/249 MM-lambda 1/22 9/31 13/42 16/25 5/7 6/17 23/95 27/49 50/144

Mutations/3NTPS FL-kappa 0/0 6/13 14/33 17/29 6/20 16/29 20/46 39/78 59/124 FL-heavy 6/36 5/28 41/80 16/25 66/99 52/144 82/124 134/268 MM-kappa 4/6 14/29 42/120 35/58 20/38 28/48 60/155 83/144 143/299 MM-lambda 1/6 15/31 18/75 13/37 8/23 12/37 34/112 33/97 67/209

3NTPS/Mutated FL-kappa 0/39 5/66 17/113 12/73 6/25 14/44 22/218 32/142 54/360 FL-heavy 7/66 4/39 39/153 17/27 57/107 50/256 76/134 126/390 MM-kappa 4/42 18/72 40/123 84/92 11/30 23/49 62/137 68/171 130/308 MM-lambda 1/34 12/29 16/57 12/32 8/13 9/20 29/120 29/65 58/185

Mutations/RGYW FL-kappa 0/4 1/12 4/38 8/28 0/20 6/27 5/54 14/75 19/129 FL-heavy 3/43 7/29 11/83 12/25 27/101 21/155 39/126 60/281 MM-kappa 0/9 10/30 5/42 26/57 3/34 10/48 23/149 39/139 62/287 MM-lambda 0/9 4/32 9/76 9/35 3/23 9/40 13/117 21/98 34/215

RGYW/Mutated FL-kappa 0/1 1/30 4/30 8/28 0/4 6/18 5/61 14/50 19/111 FL-heavy 3/30 5/21 10/21 8/15 18/34 18/72 26/49 44/121 MM-kappa 0/2 10/33 13/38 21/33 2/10 8/16 23/72 31/59 54/131 MM-lambda 0/7 4/16 8/24 8/14 3/5 4/10 12/42 15/27 27/72

Mutations/WRCY FL-kappa 1/4 4/12 5/38 5/28 2/20 4/27 10/54 11/75 21/129 FL-heavy 7/43 0/29 15/83 9/25 31/101 22/155 40/126 62/281 MM-kappa 0/9 12/30 1/9 13/57 5/34 8/48 24/149 26/139 55/287 MM-lambda 1/9 8/32 7/76 11/35 2/23 4/40 16/117 17/98 33/215

WRCY/Mutated FL-kappa 1/9 3/33 4/36 5/15 2/6 4/10 8/78 11/31 19/109 FL-heavy 7/26 0/1 14/32 9/13 18/18 21/59 26/31 44/90 MM-kappa 0/9 11/39 11/35 12/20 4/7 4/8 19/83 20/35 39/118 MM-lambda 1/17 5/15 5/18 8/11 2/4 2/7 11/50 12/22 23/72

Mutations/4NTPs, mutations found in tetranucleotides, Mutations/3NTPs, mutations found in trinucleotides, 4NTPs/Mutated, number of tetran- ucleotides mutated; 3NTPs/Mutated, number of trinucleotides mutated, Mutations/RGYW or WRCY, mutations found in RGYW or WRCY; RGYW or WRCY/mutated, number of mutated RGYW or WRCY motifs. incidence of mutations was dependent on the actual sequence V␬ vs FL-V␬. Furthermore, we compared the incidence of and location of the nucleotide motifs in the CDRs and frame- mutated tetranucleotides and trinucleotides in the same sub- work regions (FWRs). To this purpose, we examined eight groups of sequences. We then examined possible differences highly mutable trinucleotides (AGC, GCT, ATT, AAT, TAC, in the frequency with which different tetranucleotides (RGYW GTA, AGT and AGA),13 most of which are part of the or WRCY) were targeted by the somatic hypermutation RGYW/WRCY motif. mechanism.

Statistical analyses Results

Statistical analyses of the distribution of mutations in tri-and The absolute numbers of mutations in tetra-and tri-nucleo- tetra-nucleotides were carried-out by Student’s t-test and tides and their distributions in each CDR, FWR, entire V ␹2 paired . We compared the incidence of mutations in VH vs sequence, RGYW/WRCY motifs, as well as the numbers of V␬ sequences in FL, V␬ vs V␭ sequences in MM, and MM- mutated tetra-and tri-nucleotidesand their distributions in

Leukemia Hypermutation in Ig V genes of FL and MM C Belessi et al 1774 Table 2 Comparisons of the ratios of mutations in tetranucleot- the number of mutated RGYW tetranucleotides in MM-V␬ vs ides (␹2) ␬ Ͻ ␬ Ͻ FL-V (P 0.001) and FL-VH vs FL-V (P 0.001) (Table 3 and Figure 1); (4) the number of mutated WRCY tetranucleot- Total CDRs FWRs ␬ ␬ = ␬ ides in MM-V vs FL-V (P 0.006) and FL-VH vs FL-V (P Ͻ 0.001) (Table 3 and Figure 1); and (5) the targeting of ␹2 Mutations/4NTPS RGYW was significantly higher when compared to WRCY FL␬ vs MM␬ NSD NSD NSD = MM␬ vs MM␭ P = 0.01 P = 0.03 NSD tetranucleotides in the CDRs of FL-VH genes (P 0.004) (not FL␬ vs FLH NSD P = 0.02 NSD shown).

Mutations/RGYW ␹2 FL␬ vs MM␬ NSD NSD NSD Trinucleotide hotspots MM␬ vs MM␭ NSD NSD NSD FL␬ vs FLH NSD P = 0.05 NSD Similar results (regarding statistical significance) were Mutations/WRCY ␹2 obtained when undertaking intergroup comparisons for FL␬ vs MM␬ NSD NSD NSD trinucleotides (Table 4 and Figure 2). MM␬ vs MM␭ NSD NSD NSD For more detailed presentation of all these results see also FL␬ vs FLH NSD NSD NSD Tables 1–4 and Figures 1 and 2.

NSD, no significant difference. Discussion

each CDR, FWR, entire V sequence, RGYW/WRCY motifs are It has been demonstrated that in V␬ genes from normal per- demonstrated in Table 1. In general, no statistically significant ipheral B cells carrying non-productive V␬-J␬ rearrangements, differences were found when comparing the number of tetran- each RGYW tetranucleotide and its corresponding WRCY ucleotides that might serve as mutational hotspots in any kind ␬ ␬ ␭ inverse repeat contained mutations at comparable fre- of sequence examined (FL-VH, FL-V , MM-V , and MM-V ). quencies, with mutations in G and C being significantly more However, in FL-VH genes the number of RGYW tetranucleot- prevalent. Moreover, mutations in codons contained within ides was significantly higher than the number of WRCY tetran- Ͻ ␬ the RGYW/WRCY motifs were significantly more frequent in ucleotides (P 0.001). Regarding ‘mutation load’, MM-V the CDRs than in the FWRs of productive vs non-productive sequences were more heavily mutated than FL-V␬ sequences; 13 ␬ rearrangements. These results were interpreted as strong a similar finding was noted between FL-VH and FL-V indicators that the hypermutation mechanism targets the over- sequences. represented RGYW motifs in V␬ genes on both DNA strands Statistically significant differences were obtained when and that the resulting replacement mutations are preferentially comparing tetranucleotide and trinucleotide hotspots, as selected in the productive repertoire. shown below. In the present study, we examined the targeting of somatic hypermutation in the clonogenic Ig-V sequences of FL and MM, B cell tumors corresponding to advanced (post-immune) Tetranucleotide hotspots differentiation stages in B cell ontogeny. FL is a germinal center (GC) B cell malignancy.14 The neoplastic cells can be (1) The ratio of mutations in tetranucleotides (RGYW, WRCY, + ␬ considered as counterparts to intra-GC B cells undergoing RGYW WRCY)/mutations in the whole V sequence in MM-V active selection by antigen; they exhibit ongoing Ig gene hyp- vs MM-V␭ (P = 0.01) (Table 2); (2) the total number of mutated ␬ ␬ Ͻ ermutation and bear the t(14;18) chromosomal translocation, tetranucleotides in MM-V vs FL-V (P 0.01) (Table 3); (3) which leads to the formation of the hybrid bcl-2/IgH gene.15 ␬ Sequence analysis of rearranged VH and V genes in the FL Table 3 Comparisons of the frequences of mutated tetranucleo- cases under study has demonstrated that VH genes were tides (␹2) mostly hypermutated, whereas V␬ genes markedly differed regarding both the mutational load and the distribution of Total CDRs FWRs mutations.10 It appears that the potential contribution of FL- V␬ genes in antigen selection of the clonogenic B cells is less 2 12,14 4NTPS Mutated ␹ important than that of VH genes. Similar conclusions have FL␬ vs MM␬ P Ͻ 0.001 P = 0.002 P Ͻ 0.001 been reached from single-cell studies in the normal peripheral MM Ͼ FL B cell repertoire, indicating a more limited mutational load MM␬ vs MM␭ NSD NSD NSD ␬ ␬ Ͻ Ͼ ␬ Ͻ Ͻ both in the expressed as well as non-functional V genes com- FL vs FLH P 0.001 H P 0.001 P 0.001 + − 16 pared to their partner VH genes in IgM /CD5 B cells. The ␬ RGYW Mutated ␹2 fact that clonogenic V genes are frequently unmutated indi- FL␬ vs MM␬ P Ͻ 0.001 P = 0.006 P Ͻ 0.001 cates that the somatic hypermutation machinery might have MM Ͼ FL M Ͼ F ceased to operate in the V␬ locus at the time when neoplastic MM␬ vs MM␭ NSD NSD NSD transformation had occurred. In the majority of FL cases ␬ Ͻ Ͼ ␬ = = FL vs FLH P 0.001 H P 0.01 P 0.006 included in our study (7/10; 70%), significant clustering of ␬ ␹2 mutations was observed in the CDRs of either VH or V WRCY Mutated 10 FL␬ vs MM␬ P = 0.006 P = 0.05 P = 0.03 genes; thus, it is reasonable to argue that a complementarity MM␬ vs MM␭ NSD NSD NSD imprint of antigen selection witnessed by the clonogenic VH ␬ Ͻ Ͼ ␬ Ͻ Ͼ ␬ Ͻ Ͼ ␬ FL vs FLH P 0.001 H P 0.001 H P 0.001 H and VL sequences might constitute an important event in the pathogenesis of FL.14 No physiological analog to this phenom- NSD, no significant difference. enon has been observed; however, similar observations were

Leukemia Hypermutation in Ig V genes of FL and MM C Belessi et al 1775

␬ ␭ ␬ Figure 1 Percentage of mutations within MM-V /V and FL-VH/V sequences in RGYW (upper left), WRCY (lower left) tetranucleotide hotspots, as well as percentages of RGYW (upper right) and WRCY (lower right) mutated.

Table 4 Comparison of mutation ratios and incidence in trinucleo- region gene rearrangements in MM indicates that, before tide hotspots transformation, the malignant stem cell (whose exact origin remains elusive) has already undergone antigen selection with Total CDRs FWRs consistent lack of intraclonal diversification.19,20 In our series, analysis of LC V region genes has revealed somatic hypermut- Mutations/3NTPS ␹2 ␬ ␬ ation of almost the same magnitude as that reported by others FL vs MM NSD NSD NSD 11,17,21 MM␬ vs MM␭ P Ͻ 0.001 P Ͻ 0.001 NSD for VH genes. This finding is in contrast to our obser- FL␬ vs FLH NSD P = 0.02 NSD vations in FL V␬ genes and offers more direct evidence that MM originates from transformation of late post-GC B cell 3NTPS Mutated ␹2 clones.22 Furthermore, it indicates that hypermutation of V ␬ ␬ Ͻ = Ͻ L FL vs MM P 0.001 P 0.002 P 0.001 genes might serve as a surrogate marker of assigning discrete MM Ͼ FL MM␬ vs MM␭ NSD NSD NSD developmental stages regarding GC and post-GC B cell devel- 23 FL␬ vs FLH P Ͻ 0.001 H Ͼ ␬ P Ͻ 0.001 P = 0.005 opment. Our findings regarding the distribution and possible targeting of somatic mutations in rearranged IgV genes of FL and MM conform well with the aforementioned data derived from normal peripheral B cells; for example, the correspond- 17 ing malignant B cells carrying productive V␬-J␬ rearrange- made in an analysis of clonogenic VH and VL genes in MM. Moreover, as mentioned earlier, FL are characterized by ongo- ments and having experienced antigen selection generally ing somatic mutations of their Ig V genes;5 however, as the exhibit an increased incidence of RGYW targeting in CDRs vs present analysis was restricted to diagnostic samples no infor- FWRs.13 The differences observed in favor of tetranucleotide ␬ ␬ mation can be gathered regarding ongoing intraclonal diversi- (RGYW and WRCY) targeting in FL-VH vs FL-V and MM-V ␬ ␬ fication of FL Ig VH and V genes and whether these ongoing vs FL-V may implicate differences in the evolution of somatic mutations are targeted at specific hotspots. It would be rather hypermutation coupled with selection in memory (post-GC) interesting to examine this issue in future studies. vs GC B cells.24–31 Interestingly, this pattern of somatic hyper- Multiple myeloma represents a malignancy of the immune mutation targeting to tetranucletides (H Ͼ ␬ Ͼ ␭) as well as system characterized by the presence of a continuously differ- the trend for strand-biased targeting only in the CDRs of FL-

entiating population of mainly late stage B cells giving rise to VH genes (RGYW being significantly more mutated than 18 plasma cells. The analysis of variable heavy chain (VH) WRCY) and not in LC V genes recapitulates the temporal pat-

Leukemia Hypermutation in Ig V genes of FL and MM C Belessi et al 1776

␬ ␭ ␬ Figure 2 Percentage of mutations within MM-V /V and FL-VH/V sequences in tetranucleotide hotspots in total (upper left) and in trinucleo- tide hotspots in total (lower left), as well as percentages of tetranucletide hotspots in total (upper right) and trinucleotide hotspots in total (lower right) mutated.

tern of Ig gene rearrangements in early B cell ontogeny, where leads to a redistribution of mutations away from hotspots, thus Ig HC genes rearrange first, to be followed by ␬ and then ␭ equilibrating hotspot vs non-hotspot targeting of somatic hyp- LC genes;32 however, exceptions to this generally occurring ermutation. Therefore, in the absence of MMR genes, dimin- pattern of ordered light chain gene rearrangements are known ished mutation accumulation and increased hotspot focusing to occur (instead of ␬ preceding ␭, in a minority of cases the would be anticipated. MMR gene inactivation and deficiency, ␭ light chain locus is targeted first by the ‘recombinase’ leading to the replication-error phenotype, is a common event machinery).33 in B cell malignancies. With this in mind, it would be reason- In the present analysis, hotspot sequences were not always able to speculate that in some cases of FL and MM the neo- targeted by somatic hypermutation in FL and MM. Several plastic B cells might have lost their ability to correct mutations explanations can be offered for this observation: (1) other introduced in classic hotspots; conversely, in other cases with unrecognized motifs may be targets of somatic hypermutation; intact MMR function, the hotspot to non-hotspot targeting of (2) despite the fact that the cell of origin in FL and MM has somatic hypermutation is balanced. been assigned at an ontogenetic stage postulated to be subject V␭ genes in MM appear to be targets of somatic hypermut- to antigen selection in GCs, it could be argued that many ation, albeit at sequences outside the recognized hotspots for ␬ ␬ ␭ mutations introduced in V genes would not favor selection VH and V genes. This might implicate that V genes are tar- by antigen under normal conditions;34 neoplastic transform- geted by the somatic hypermutation mechanism at later devel- ation at this stage might have over-ruled this requirement for opmental stages of B cell ontogeny, where the aforementioned cell survival by rescuing these cells from apoptosis, similar to hotspots are no longer important in directing mutation what has been reported for Hodgkin’s disease.35 Therefore, targeting.37 these ‘inappropriately’ introduced mutations were fixed and The CDR3 of IgH, ie the IgH-V subregion mainly respon- propagated by the neoplastic process; (3) mismatch-repair sible for antigen binding is formed by the junctions of the

(MMR) mechanisms may play an important role in the distri- rearranged VH–D–JH gene segments. In normal B cell develop- bution of somatic mutations in GC B cells;36 thus, one may ment, the amino acid composition of the IgH CDR3 is the speculate that at an early, MMR-independent phase of somatic main determinant of positive selection.38 Formation of CDR3 hypermutation, mutations are introduced at specific hotspots region is accomplished during rearrangement of V–(D)–J genes (G/C biased); later, during a second MMR-dependent stage, with the insertion of N-nucleotides by terminal deoxynucleo- mutations are introduced in the target sequence with an A/T tide transferase (TdT), while random deletion and insertion of bias through the action of an error-prone polymerase. This bases is effected at the borders of the rearranging genes (V/J

Leukemia Hypermutation in Ig V genes of FL and MM C Belessi et al 1777 genes for light chains and V/D/J genes for heavy chains). The T. Molecular insights to the immunopathogenesis of follicular lym- phoma. Immunol Today 2000; 21: 298–305. randomly selected D gene segment of a rearranged VDJH com- plex can potentially be found in all three possible reading 15 Stamatopoulos K, Kosmas C, Belessi C, Papadaki T, Afentaki S, 39 Anagnostou D, Loukopoulos D. t(14;18) chromosomal translo- frames (RFs). However, as we have shown, particular D gene cation in follicular lymphoma: an event occurring with almost

RFs can be encountered preferentially in certain B cell equal frequency both at the D to JH and at later stages in the lymphoproliferative disorders as a result of the maturation rearrangement process of the immunoglobulin heavy chain gene status of the corresponding transformed B cell and selection locus. Br J Haematol 1997; 99: 866–872. by antigen.40 The similarly restricted pattern of D gene RF 16 Klein U, Goossens T, Fischer M, Kanzler H, Braeuninger A, Rajew- usage (predominantly RF2 and RF3) in both functional IgH sky K, Ku¨ppers R. Somatic hypermutation in normal and transfor- med human B cells. Immunol Rev 1998; 162: 261–280. junction sequences in FL and MM and non-functional bcl- 17 Sahota SS, Leo R, Hamblin TJ, Stevenson FK. Myeloma VL and VH 2/DJH junction sequences in FL suggests that selection forces sequences reveal a complementary imprint of antigen selection in might affect Ig genes while still in the process of active recom- tumor cells. Blood 1997; 89: 219–226. bination (DJH complexes); in this context, it has been pro- 18 Kosmas C, Stamatopoulos K, Stavroyianni N, Zoi K, Belessi C, posed that pre-B cell clones are submitted to an antigen- Viniou N, Kollia P, Yataganas X. Origin and diversification of the independent initial selective pressure, whereby D␮ chains clonogenic cell in multiple myeloma: lessons from the immuno- would be merely selected for their ability to interact with sur- globulin repertoire. Leukemia 2000; 14: 1718–1726. 41 19 Bakkus MHC, Van Riet I, Van Camp B, Thielemans K. Evidence rogate light chains. This observation becomes important in that the clonogenic cell in multiple myeloma originates from a indicating that even at early stages of B cell ontogeny, where pre-switched but somatically mutated B cell. Br J Haematol 1994; somatic hypermutation is not operating, selective forces might 87: 68–74. affect the conformation of V region and prime certain B cell 20 Vescio RA, Cao J, Hong CH, Lee JC, Wu CH, Der-Danielian M, clones to fine-tune their specificity for antigen later, in Wu V, Newman R, Lichtenstein AK, Berenson JR. Myeloma Ig the germinal center, under the influence of somatic heavy chain V region sequences reveal prior antigenic selection and marked somatic mutation but no intraclonal diversity. J Immu- hypermutation. nol 1995; 155: 2487–2497. 21 Wagner SD, Martinelli V, Luzzatto L. Similar patterns of V␬ gene usage but different degrees of somatic mutation in hairy cell leuke- References mia, prolymphocytic leukemia, Waldenstro¨m’s macroglobuline- mia, and myeloma. Blood 1994; 83: 3647–3653. 22 Kosmas C, Stamatopoulos K, Stavroyianni N, Belessi C, Viniou 1 Tonegawa S. Somatic generation of antibody diversity. Nature N, Yataganas X. Molecular analysis of immunoglobulin genes in 1983; 302: 575–581. multiple myeloma. Leuk Lymphoma 1999; 33: 253–263. 2 Grawunder U, West RB, Lieber MR. Antigen receptor gene 23 Kuppers R, Klein U, Hansmann M-L, Rajewsky K. Cellular origin rearrangement. Curr Opin Immunol 1998; 10: 172–180. ¨ of human B-cell lymphomas. N Engl J Med 1999; 341: 1520– 3 Rajewsky K. Clonal selection and learning in the antibody system. Nature 1996; 381: 751–758. 1529. 4 Storb U. Progress in understanding the mechanism and conse- 24 Kelsoe G. V(D)J hypermutation and receptor revision: coloring quences of somatic hypermutation. Immunol Rev 1998; 162:5– outside the lines. Curr Opin Immunol 1999; 11: 70–75. 11. 25 Milstein C, Neuberger MS, Staden R. Both DNA strands of anti- 5 Stevenson F, Sahota S, Zhu D, Ottensmeir C, Chapman C, Oscier body genes are hypermutation targets. Proc Natl Acad Sci USA D, Hamblin T. Insight into the origin and clonal history of B-cell 1998; 95: 8791–8794. tumors as revealed by analysis of immunoglobulin variable region 26 Nakamura N, Kuze T, Hashimoto Y. Hara V, Hoshi S, Sasaki Y, genes. Immunol Rev 1998; 162: 247–259. Shirakawa A, Seto M, Abel M. Analysis of the immunoglobulin 6 Chang B, Casali P. The CDR1 sequences of a major proportion of heavy chain gene variable region of CD5-positive and -negative human germline IgVH genes are inherently susceptible to amino diffuse large B cell lymphoma. Leukemia 2001; 16: 452–457. acid replacement. Immunol Today 1994; 15: 367–373. 27 Capello D, Fais F, Vivenza D, Migliaretti G, Chiorazzi N, Gaidano 7 Betz AG, Rada C, Pannell R, Milstein C, Neuberger MS. Passenger G, Ferrarini M. Identification of three subgroups of B cell chronic transgenes reveal intrinsic specificity of the antibody hypermut- lymphocytic leukemia based upon mutations of BCL-6 and IgV ation mechanism: clustering, polarity, and specific hot spots. Proc genes. Leukemia 2000; 14: 811–815. Natl Acad Sci USA 1993; 90: 2385–2388. 28 Fais F, Gaidano G, Capello D, Gloghini A, Ghiotto F, Roncella S, 8 Wagner SD, Milstein C, Neuberger MS. Codon bias targets Carbone A, Chiorazzi N, Ferrarini M. Immunoglobulin V region mutation. Nature 1995; 376: 732. gene use and structure suggest antigen selection in AIDS-related 9 Neuberger NS, Ehrenstein MR, Kllx N, Jolly CJ, Ye´lamos J, Rada primary effusion lymphomas. Leukemia 1999; 13: 1093–1099. C, Milstein C. Monitoring and interpreting the intrinsic features of 29 Driessen A, Tierens A, Ectors N, Stul M, Pittaluga S, Geboes K, somatic hypermutation. Immunol Rev 1998; 162: 107–116. Delabie J, De Wolf-Peeters C. Primary diffuse large B cell lym- 10 Stamatopoulos K, Kosmas C, Papadaki T, Pouliou E, Belessi C, phoma of the stomach: analysis of somatic mutations in the Afendaki S, Anagnostou D, Loukopoulos D, Follicular lymphoma rearranged immunoglobulin heavy chain variable genes indicates immunoglobulin ␬ light chains are affected by the antigen selec- antigen selection. Leukemia 1999; 13: 1085–1092. tion process, but to a lesser degree than their partner heavy chains. 30 Kosmas C, Stamatopoulos K, Immunoglobulin light chain variable Br J Haematol 1997; 96: 132–146. region genes in multiple myeloma. Leukemia 1999; 13: 827–830. 11 Kosmas C, Viniou NA, Stamatopoulos K, Courtenay-Luck NS, 31 Kon S, Sasamori T, Kasai K, Yamano H, Endo T, Kon H, Kikuchi K. Papadaki T, Kollia P, Paterakis G, Anagnostou D, Yataganas X, Ongoing somatic mutations of the immunoglobulin gene in MALT Loukopoulos D. Analysis of ␬ light chain variable region in mul- lymphoma with widespread MLP type polypoid lesions. Leukemia tiple myeloma. Br J Haematol 1996; 94: 306–317. 1998; 12: 1495–1497. 12 Kosmas C, Stamatopoulos K, Papadaki T, Belessi C, Yataganas X, 32 Korsmeyer SJ, Hieter PA, Ravetch JV, Poplack DG, Waldmann TA, Anagnostou D, Loukopoulos D. Somatic hypermutation of Leder P. Developmental hierarchy of immunoglobulin gene immunoglobulin genes: focus on follicular lymphoma and mul- rearrangements in human leukemic pre-B-cells. Proc Natl Acad tiple myeloma. Immunol Rev 1998; 162: 281–292. Sci USA 1981; 78: 7096–7100. 13 Do¨rner T, Foster SJ, Brezinschek H-P, Lipsky PE. Analysis of the 33 Pauza ME, Rehmann JA, LeBien TW. Unusual patterns of immuno- targeting of the hypermutational machinery and the impact of sub- globulin gene rearrangement and expression during human B-cell sequent selection on the distribution of nucleotide changes in ontogeny: human B-cells can simultaneously express cell surface ␬ ␭ human VHDJH rearrangements. Immunol Rev 1998; 162: 161– and light chains. J Exp Med 1993; 178: 139–149. 171. 34 Nossal GJV. Negative selection of lymphocytes. Cell 1994; 76: 14 Stamatopoulos K, Kosmas C, Belessi C, Kyriazopoulos P, Papadaki 229–239.

Leukemia Hypermutation in Ig V genes of FL and MM C Belessi et al 1778 35 Kanzler H, Ku¨ppers R, Hansmann M-L, Rajewsky K. Hodgkin’s capacity for binding ligands. Proteins 1990; 7: 112–124. and Reed–Sternberg cells in Hodgkin’s disease represent the out- 39 Raaphorst FM, Raman CS, Nall BT, Teale JM. Molecular mech- growth of a dominant tumor clone derived from (crippled) germi- anisms governing reading frame choice of immunoglobulin diver- nal center B cells. J Exp Med 1996; 184: 1495–1505. sity genes. Immunol Today 1997; 18: 37–43. 36 Rada C, Ehrenstein MR, Neuberger MS, Milstein C. Hot spot focus- 40 Stamatopoulos K, Kosmas C, Stavioyianni N, Belessi C, Papadaki ing of somatic hypermutation in MSH2-deficient mice suggests T. Selection of immunoglobulin diversity gene reading frames in two stages of mutational targeting. Immunity 1998; 9: 135–141. B-cell lymphoproliferative disorders. Leukemia 1999; 13:601– 37 Farner NL, Do¨rner T, Lipsky PE. Molecular mechanisms and selec- 604. tion influence the generation of the human V␭–J␭ gene repertoire. 41 Millili M, Schiff C, Fougereau M, Tonnelle C. The VDJ repertoire J Immunol 1999; 162: 2137–2145. expressed in human pre B cells reflects the selection of bona fide 38 Padlan EA. On the nature of antibody combining sites: unusual heavy chains. Eur J Immunol 1996; 26: 63–69. structural featutres that may confer on these sites an enhanced

Leukemia