anti-Similarities and Differences Between the Light and Heavy Chain Ig Variable Region Gene Repertoires in Chronic Lymphocytic Leukemia

Analyses of Ig V H DJ H rearrangements expressed by B-CLL cells have provided insights into the antigen receptor repertoire of B-CLL cells and the maturation stages of B-lymphocytes that give rise to this disease. However, less information is available about the L chain V gene segments utilized by B-CLL cells and to what extent their characteristics resemble those of the H chain. We analyzed the V L and J L gene segments of 206 B-CLL patients, paying particular attention to frequency of use and association, mutation status, and LCDR3 characteristics. Approximately 40% of B-CLL cases express V L genes that differ significantly from their germline counterparts. Certain genes were virtually always mutated and others virtually never. In addition, preferential pairing of specific V L and J L segments was found. These findings are reminiscent of the expressed VH repertoire in B-CLL. However unlike the V H repertoire, V L gene use was not significantly different than that of normal B-lymphocytes. In addition, V κ genes that lie more upstream on the germline locus were less frequently mutated than those at the 3 ′ end of the locus; this was not the case for V λ genes and is not for V H genes. These similarities and differences between the IgH and IgL V gene repertoires expressed in B-CLL suggest some novel features while also reinforcing concepts derived from studies of the IgH repertoire.


INTRODUCTION
Immunoglobulin (Ig) variable (V) domains are the components of the B-cell antigen receptor (BCR) that interact with antigen. Understanding the gene segments that encode these domains can provide indirect information about the structure of the BCR. In addition, somatic changes that occur in these genes can suggest clues regarding the maturational history of a B lymphocyte.
These principles have been of special value in understanding the biology of the B lymphocytes that become leukemic in B-cell chronic lymphocytic leukemia (B-CLL). For example, analyses of V H DJ H rearrangements expressed by these clones (1)(2)(3) has led to the recognition that B-CLL cases segregate into subgroups based on the presence or absence of mutations in V H genes, i.e., mutated and unmutated B-CLL, respectively (4,5). This division has considerable prognostic significance (6,7). Patients in the Ig V H mutated subgroup have a relatively benign clinical course. These individuals can live for many years after diagnosis (10-25 years), usually do not require therapy, and often die with the disease, not because of it. In contrast, patients in the unmutated subgroup follow a much more aggressive clinical course; these people have a median survival of less than 8 years, despite extensive therapeutic efforts which may quell but do not cure the disease. B-CLL remains incurable.
Furthermore, these studies suggest that the leukemic cells from both the mutated and unmutated subgroups are anti-

Similarities and Differences Between the Light and Heavy Chain Ig Variable Region Gene Repertoires in Chronic Lymphocytic Leukemia
Analyses of Ig V H DJ H rearrangements expressed by B-CLL cells have provided insights into the antigen receptor repertoire of B-CLL cells and the maturation stages of B-lymphocytes that give rise to this disease. However, less information is available about the L chain V gene segments utilized by B-CLL cells and to what extent their characteristics resemble those of the H chain. We analyzed the V L and J L gene segments of 206 B-CLL patients, paying particular attention to frequency of use and association, mutation status, and LCDR3 characteristics. Approximately 40% of B-CLL cases express V L genes that differ significantly from their germline counterparts. Certain genes were virtually always mutated and others virtually never. In addition, preferential pairing of specific V L and J L segments was found. These findings are reminiscent of the expressed VH repertoire in B-CLL. However unlike the V H repertoire, V L gene use was not significantly different than that of normal B-lymphocytes. In addition, Vκ genes that lie more upstream on the germline locus were less frequently mutated than those at the 3′ end of the locus; this was not the case for Vλ genes and is not for V H genes. These similarities and differences between the IgH and IgL V gene repertoires expressed in B-CLL suggest some novel features while also reinforcing concepts derived from studies of the IgH repertoire. Online address: http://www.molmed.org doi: 10.2119/2006-00080. Ghiotto gen-experienced B-lymphocytes. Observations on the surface phenotypes (8) and the gene expression profiles (9,10) of the leukemic cells corroborate this notion. Finally, biased use of V H , D, and J H gene segments (5,7,(11)(12)(13) and selective combinatorial associations (14)(15)(16)(17)(18)(19) suggest that the antigenic epitopes responsible for this activated and antigen-experienced/memory state are limited in nature. An alternative, not mutually exclusive explanation, is that the normal B-cells from which B-CLL cells derive are markedly restricted in their antigenbinding repertoire, either genetically or due to prior antigen selection (1,3,16,17).
Thus, considerable basic as well as clinical information has been gleaned from studying the rearranged V H DJ H segments that code for the V domains of the Ig H chain in these leukemic cells. In contrast, less information is currently available about the gene segments that make up the V domains of the Ig L chains of B-CLL cells and the extent to which the characteristics of these segments resemble those of the H chain (20). Therefore, it is not resolved if conclusions about the immunobiology of B-CLL cells that were derived from H chain data are recapitulated by the rearranged V L J L segments. In this study, we analyzed the V L and J L gene segments expressed by a large cohort of B-CLL patients, focusing on the frequency of use and association, mutation status, and characteristics of the 3rd complementarity determining region (CDR3) that is critical for antigen-binding (21,22). These analyses reveal similarities as well as some differences in these features between the V region H and L chain repertoires expressed in B-CLL.

B-CLL Samples
We analyzed the DNA sequences of V L J L rearrangements of 206 patients with B-CLL; 179 of these patients had expansions of IgM + /CD5 + /CD19 + B cells and 27 patients displayed expansions of CD5 + / CD19 + B cells expressing smIgG + or smIgA + . DNA sequences for some of these cases have been described (5,16,23). PBMC, obtained from heparinized venous blood by density gradient centrifugation (Ficoll-Paque; Pharmacia LKB Biotechnology, Piscataway, NJ), were used immediately or cryopreserved with a programmable cell-freezing machine (CryoMed, Mt. Clemens, MI) prior to being thawed and analyzed. cDNA prepared from samples were screened for expression of a dominant V L family (representing that of the B-CLL clone) by standard PCR. In this study we included only B-CLL cells that exhibited allelic exclusion.

Preparation of RNA and cDNA Synthesis
Total RNA was isolated from fresh or cryopreserved PBMC using Ultraspec RNA (Biotecx Laboratories, Houston, TX) according to the manufacturer's instructions. One µg of RNA was reverse transcribed using 200U M-MLV reverse transcriptase (GIBCO BRL, Life Technologies, Grand Island, NY), 1U of RNase inhibitor (Eppendorf, Hamburg, Germany), as previously described (5).

Ig V L J L Gene Sequencing and Analysis
V L J L sequences were determined as reported previously (16). Sequences were compared with the V BASE sequence directory (24) using MacVector software, version 7.0 (Accelrys, San Diego, CA), to GenBank, and to the international Im-MunoGeneTics information system® http://imgt.cines.fr (Initiator and coordinator: Marie-Paule Lefranc, Montpellier, France; ref. (25)). In those instances where > 1% mutation was found in an expressed V L gene, the algorithm of Chang and Casali (26) was employed to determine the extent to which "antigenselection" of the replacement (R) mutations had occurred, taking into account the inherent susceptibility of CDR to R mutations. The expected number of R mutations in CDR and FR was calculated using the formula R = n × CDR Rf (or FR Rf) × CDRrel (or FRrel) where n is the total number of observed mutations, Rf is the R frequency inherent to the CDR or FR, and CDRrel and FRrel are the relative sizes of these segments. A binomial probability model was used to evaluate whether the excess of R mutations in CDR or the scarcity in FR was due to chance (26).

Analyses of LCDR3 Rearrangements
LCDR3 length was determined by counting the number of amino acids (aa) immediately following the conserved cystine (C) at position 88 at the end of FR3 to the aa preceding position 98 at the beginning of FR4 (a conserved phenylalanine (F) in all JL segments). LCDR3 charge, as defined by an estimated pI, was determined using the MacVector software program (version 7.0).

Statistical Analyses
Analyses focused primarily on descriptive statistics (summaries using means, medians, standard deviations, proportions). Additional analyses examined associations between study groups (B-CLL vs. normal subjects) and V L isotypes (κ vs. λ), C H isotypes (IgM + vs. non-IgM + ), V H mutation status, and other categorical variables using the Fisher's Exact Test. The standard goodness-of-fit test was used to determine whether specific Vκ-Jκ and Vλ-Jλ pairings were more frequently encountered than others. The Mann-Whitney test was used to compare LCDR3 length and charge between specific group comparisons.

κ and λ L Chain Use in B-CLL Cells
The distribution of κ and λ chain use in normal, polyclonal B-cell populations is 2:1 (27). Therefore, we analyzed the V L gene sequences expressed by 206 B-CLL clones (179 IgM + and 25 IgG + and 2 IgA + ) to determine if this was the case in the B cells transformed in this disease (Supplemental Tables S1, S2). In 67.9% (140/206) of the cases, the leukemic cells expressed a Vκ gene and in 32.0% (66/206) a Vλ gene. This distribution was similar for IgM + (66.5% κ + and 33.5% λ + ) and non-IgM + (77.8% κ and 22.2% λ) cases. Thus, the ratio of κ and λ chains in B-CLL resembles that of normal B lymphocytes.

V L Gene Family Use
Among the 140 κ-expressing samples, Vκ genes were derived from families I, II, III, IV, and VI (Table 1) in the following order of frequency: VκI (52.1%) > VκIII (25.7%) > VκII (16.4%) > VκIV (5.0%) > VκVI (0.7%). The order of Vκ family distribution and their relative frequencies were unchanged when the cases were divided into subgroups based on C H isotype. In addition, there was no significant difference in the distribution and frequency of Vκ family use in IgM + B-CLL cases compared with the reported repertoires of normal IgM + CD5 + and IgM + CD5 -B cells (Table 1) (28,29). A comparison between non-IgM + B-CLL cases and normal non-IgM + CD5 + and CD5 -B cells could not be performed because data on the latter 2 control populations are lacking in the literature.

Use of Specific Vκ and Vλ Genes
IgV use among normal B lymphocytes is not stochastic; rather it is skewed by genetic and environmental pressures (33)(34)(35)(36). The distribution of individual Vκ genes of IgM + B-CLL cases resembled that reported in normal CD5 + and CD5 -IgM + B cells (data not shown) (28,29). A similar comparison of specific Vλ gene expression was not possible because of the lack of data reported for normal control subsets.

Number and Location of VL Gene Mutations
When mature B cells encounter antigen, they can undergo the somatic hyper- mutation process that alters the structure of Ig H and L V region genes and possibly the protein structure of the BCR [reviewed in (37)]. This is especially frequent if the antigen bound elicits T-cell help (38). Thus the presence of IgV mutations indicates antigenic experience as well as implies maturational pathway. We analyzed the number, type, and location of somatic changes in expressed V L J L of our B-CLL cohort. A difference of 2% or more from the most similar germline gene was taken as the cut-off point to define a sequence as mutated (4). Approximately 42% (87/206) of B-CLL V L genes differed from the most similar germline gene by 2% or more (Table 3). C H isotypeswitched cases were more often mutated (63%) than IgM + cases (39.1%; P = 0.02). When Vκ-and Vλ-expressing cases were analyzed separately, 43.6% of the former and 39.4% of the latter differed by ≥ 2% from the germline counterpart. Many more isotype-switched Vλ-expressing cases were mutated (83.3%) than IgM + Vλ-expressing cases (35.0%; Table 3; The Vκ families differed in mutation frequency with a distribution of VκIV > VκI > VκIII > VκII (Tables S1,S2). In addition, certain Vκ genes displayed more mutations than others (Table 4). IGKV1-5 and 3-20 exhibited significant levels of muta-tion (80%, 8/10 cases and 63.6%, 12/19 cases, respectively). In contrast, other genes were rarely mutated. For example, in every instance (8/8) IGKV2-28 was > 98% similar to the germline sequence; all of these cases expressed IgM. Likewise, IGKV1-33, which was also found only among IgM + cases, and IGKV1-39 were very similar to their germline counterparts (9/10, 90%, and 21/25, 84%, respec-tively), even though 42.5% (31/73) of the VκI-expressing cases were mutated. Of Vλ-expressing cases, 85.7% (12/14) of those using IGLV3-21 were minimally divergent from the germline sequence (Tables 4,S1,S2); all these cases were IgM + .
Finally, most of the Vκ genes that remained unmutated were positioned considerably upstream on the Vκ locus (Figure S1). Specifically, 46.5% (27/58) of the   Vκ genes located at the 3′ end of the locus (within the interval from IGKV4-1 to 3-20) were unmutated, whereas 71.7% (43/60) of the Vκ genes 5′ of IGVK3-20 (starting at 6-21 and considering also the duplicated portion of the locus) were unmutated. In contrast, unmutated and mutated Vλ genes were distributed uniformly along the Vλ locus.

Types of VL Mutations
BCRs that have been selected by antigen often display a higher frequency of R mutations in CDRs and/or a lower frequency of such mutations in FRs (39,40). Based on these considerations, ~50% of both κ-expressing and λ-expressing cases demonstrated evidence of antigen selection (Tables S1,S2).
Among the IgMκ + mutated cases (Table S1), 40.1% (20/49) exhibited either a significantly increased frequency of R mutations in CDR (n = 1) or more often a significantly decreased frequency of R mutations in FR (n = 12) and 9 cases displayed mutations patterns in both the CDR and FR that were consistent with antigen selection. Of the IgMλ + mutated cases (Table S1), 47.6% (10/21) demonstrated evidence for antigen selection. In 6 cases, there were fewer R mutations in FR and in 2 instances more R mutations in CDR than predicted. In 2 cases, both criteria for selection were found.
Approximately 83% of the κ-expressing and 50% of λ-expressing isotype-switched cases demonstrated similar evidence for antigen selection (Table S2). Although none of the cases showed a significantly increased frequency of R mutations in CDR, 8 exhibited a significant decrease in R mutations in the FR and 3 displayed significant changes in CDR and FR.

Allelic Polymorphisms
To ensure that the differences observed were primarily the effect of somatic changes and did not reflect known polymorphic variants, V L genes encountered in our analyses were compared with the list of polymorphisms available in the IMGT and GenBank databases. In every instance, the identified differences were consistent with somatic mutations. Furthermore, the alleles most commonly used in B-CLL were the same as those identified in our normal subject V L database. Thus, allele IGKV1-39*01 was used in 100% of the B-CLL cases and in 91.7% of normal controls. Similarly, the frequency of use of alleles IGKV1-5*03, 3-20*01, and 3-11*01 were identical in all B-CLL and normal B-cells.

J L Gene Use
J L family use among the entire B-CLL cohort, the IgM + cases, and the non-IgM + cases are listed in Table 5. The frequency of Jκ family use was the same in each group: Jκ1 > Jκ2 > Jκ4 > Jκ3 > Jκ5. For Jλ, the order of frequency in the entire B-CLL cohort and the IgM-expressing cases was Jλ3 > Jλ2/3 > Jλ1. Of note, all the cases expressing IgG or IgA used the Jλ2/3 or Jλ3 gene segment.

V L -J L Joining
An analysis of the frequency of joining of V L gene families with J L genes indicated that there was no preferential association, either for κ (P = 0.28) or λ (P = 0.8). However, when we compared the distribution of V L -J L pairing in IgM + B-CLL with that observed in normal subjects (28-32), we did notice that Vλ2 genes paired more frequently with the Jλ3 segment in B-CLL compared with normal individuals (11.7% (7/60) versus 1.5% (4/135), P = 0.04).

LCDR3 Characteristics
The antigen-binding pocket of the BCR is a composite of both the H and L rearrangements (41). Although HCDR 1 and 2 and LCDR 1 and 2 are important contributors, the H and L CDR3s have the greatest impact on the structure of the binding site for most antigens (21,22). In B-CLL, the HCDR3 of the BCR often displays unique features that differ between the Ig V H mutated and unmutated subgroups. Therefore, we carefully examined the LCDR3 of the κand λ-expressing cases in regards to length, amino acid composition, and charge.
The average charge of LCDR3, as determined by the estimated pI value, for the κ-expressing samples was 6.5 ± 1.9 (Tables S1,S2), a value similar to those for IgM + (6.4 ± 1.9) and non-IgM + (6.9 ± 1.9) cases and cases expressing different Vκ gene families. It is also similar to the average LCDR3 pI (6.4 ± 1.9) of healthy individuals (calculated from refs. 28,29).

Pairing of the Most Commonly Encountered Vκ and Vλ Genes with Specific IgV H Genes
Because, as mentioned above, both V H DJ H and V L J L rearrangements contribute to antigen binding, we searched for selective associations of certain V L with V H genes. In fact, we found 2 examples of such associations. IGKV1-39 paired mainly with V H 1 (10/20) and V H 3 (6/20) family members in the IgM + B-CLL cases; in IgG + cases, IGKV1-39 paired almost exclusively with IGHV4-39 gene (4/5). In addition, a preferential pairing of IGVL3-21 gene with V H 3 gene family members (11/14) was identified.

DISCUSSION
In this study, we analyzed the expression of V L and J L segments in a cohort of 206 B-CLL patients, paying particular attention to [1] frequencies of utilization and association of these segments, [2] frequency, level, and location of somatic mutations, and [3] characteristics of LCDR3. We found similarities and some differences between the L chain and H chain V region repertoires of B-CLL cells (Table 6).

Gene Use
The most striking difference between the IgL and IgH repertoires is the lack of bias in V L and J L gene use. The IgH repertoire in B-CLL is characterized by a use of V H , D, and J H genes (5,7,12,42) and alleles (11,13) that differs from B cells of normal individuals. In contrast, our data indicate that expression of IgL κ and λ families and genes and J L segments mirrors that reported for the normal adult human B cell repertoire. However, Stamatopoulos recently reported skewed representation of individual IgL κ and λ genes in B-CLL (20). The reason for this discrepancy is unclear but may relate to the normal control populations used for comparison. Only, 1 clear difference between the IgL repertoire in B-CLL and normal B cells appears to exist, i.e., exclusive use of Jλ2 among λ + C H isotype-switched cases.
As might be expected from the similarity in V L and J L gene use, the LCDR3s of both the κ and λ rearrangements were similar to their counterparts in the normal B-cell repertoire. Again, this is different from the IgH repertoire, which differs in length, amino acid composition, and charge (Tables S1,S2) (5,11,13) from that of normal circulating B cells. Of note, however, certain Vλ families (Vλ2 > Vλ3 > Vλ1) do differ significantly in LCDR3 pI values. In this latter regard, the IgL and IgH B-CLL repertoires are similar because V H family-related differences exist for HCDR3 charge (V H 3 > V H 4 > V H 1) and length (for example, longer in V H 1 genes, in particular 1-69 and shorter in V H 3, in particular 3-07) (5).
However, 1 similarity in gene use does exist between the IgL and IgH repertoires in B-CLL, i.e., use of certain genes solely among IgM-expressing cases. For example, 3 of the most commonly encountered V L genes (IGKV1-33 and 2-28 of the κ repertoire and IGLV3-21 of the λ repertoire) were not found among CH isotype-switched cases. This phenomenon is reminiscent of that seen for the IGHV1-69 gene, which is rarely encountered in IgG + or IgA + B-CLL cases (16).
Interestingly, the B-CLL IgL repertoire does differ from that of normal B cells in gene family pairing. We found that Vλ2 and Jλ3 genes paired more frequently in B-CLL than in normal subjects (Tables S1,S2). At the specific gene level, in IgM + cases, IGKV1-39 associated preferentially with Jκ2 and IGLV3-21 paired often with Jλ3. However, among C H isotype-switched cases, IGKV1-39 associated most frequently with Jκ1. Coordinate association of V and J genes also occurs in IgH repertoire, where V H 1-69expressing B-CLL cells often use J H 6 and 3-07-expressing cells often use J H 4 (5,11). Our data differ somewhat from those reported recently (20), mainly by a lower percentage of IGKV4-1 and IGLV2-8 genes in our cohort.

Somatic Mutation
Another feature shared by the V genes of the H and L chain repertoires is the presence of somatic mutations, which in some instances is limited to specific genes. In approximately 42% of our B-CLL cases, the expressed V L genes differed by 2.0% or more from the most similar germline counterpart (Table 3); the level and extent of gene difference was the same for Vκ-and Vλ-expressing cases. These percentages agree with those recently reported by Stamatopoulos and co-workers (20). In addition, the frequency of mutated V L sequences was significantly higher among C H isotypeswitched cases (63%) than IgM + cases (36%; P = 0.0165); this finding resembles that of the V H repertoire in B-CLL (1-3). Of note, virtually all Vλ + cases that ex-pressed a switched C H isotype were mutated (83.3%, P = 0.01; Table 4).
The IgL and IgH gene repertoires also were similar in regards to the type and location of nucleotide differences (Tables S1,S2). Of the 87 sequences with at least 2% difference from the germline, 49.4% (43/87) exhibited evidence for antigen selection. Of these 43 cases, selection was suggested most often by a preservation of FR structure (65.2%, 28/43), followed by a preservation of FR with a change in CDR structure (27.9%, 12/43), and then solely a change in CDR structure (7.0%, 3/43). This is consistent with the pattern of R mutations in mutated V H genes in B-CLL (5,7,12,42). Finally, the definition of a somaticallymutated sequence employed here, and in prior studies of the V H repertoire in B-CLL, used a ≥ 2% difference from the most similar germline gene as an arbitrary cutoff to define "mutation." This cutoff was originally selected to account for unknown polymorphisms in the human IgV H locus (4). However, if one uses a ≥ 1% difference to assign this designation, the numbers of "mutated" sequences change minimally and insignificantly ( ≥ 1% = 47.4% mutated sequences vs. ≥ 2% = 40.7% mutated sequences). This is consistent with our finding that known polymorphisms of specific V L genes do not account appreciably for the differences reported here or for the differences in B-CLL V H gene sequences reported by others (43).

Pairing of V H and V L Segments in the BCRs of B-CLL Cells
Pairing of specific IgV H genes with V L genes was studied for the most frequently encountered Vκ (IGKV1-39) and Vλ (IGLV3-21) genes. In both instances, pairing appeared to be non-random. IGKV1-39 paired preferentially with V H 1 and V H 3 family members in IgM + cases, although no preferential coupling with a specific V H gene in these families was observed. Conversely, in IgG + cases IGKV1-39 gene almost always paired with IGHV4-39 gene. These cases represent a B-CLL subgroup with almost identical BCR structures that involve the entire VHDJH and VLJL rearrangements, including H and L CDR3s with unique amino acids at the V-(D)-J junctions (16). Similarly, IGLV3-21 gene (represented only in IgM + cases) was paired mainly with VH3 gene family members (11/14) cases) and often with IGHV3-21 (4/10). Indeed, these B-CLL cases represent another subgroup of B-CLL with remarkably similar BCR structures (15). A comprehensive analysis of IgV H and V L pairing in a large number of B-CLL cases is being prepared (Ghiotto et al., manuscript in preparation).

Concluding Remarks
What do these studies of the IgV L repertoire add to the knowledge already gleaned from studies of the IgH repertoire? The presence of significant levels of somatic mutations in the V L genes of 40% of patients confirms the conclusion drawn from V H analyses that many B-CLL cases derive from mature B-lymphocytes that have experienced antigenic stimulation at some point in their development. However, the V L data do not shed additional light on the manner in which the B-CLL cell precursors accumulated these mutations. Certainly, reactivity with a foreign antigen that elicited a classical T-cell-dependent, germinal center-mediated somatic hypermutation process may have occurred. Alternatively, a T-cell-independent process initiated by non-protein antigens, such as those expressed on the surface of certain microbes, could be responsible for the observed V L gene changes.
The V L mutation data also support the contention that B-CLL cells derive from cells selected by specific antigens. A clear bias for selection against R mutations in FR exists, because less than 50% of the "antigen selected" V L sequences involved an amino acid replacement in LCDR1 or LCDR2 (Tables S1,S2). This type of structural conservation is consistent with the need for B cells to retain an intact BCR, a principle that has been illustrated clearly in animal systems (44). Furthermore, the greater tendency to preserve FR structure, rather than altering CDR amino acid composition, can be viewed as favoring securing antigen outside of the classical binding pocket, implying that superantigen drives some B-CLL cells and their precursor B-lymphocytes. Nevertheless, the association of certain specific V, (D), and J segments (5,7,(11)(12)(13) and their selective combination in assembling H and L chain rearrangements (14)(15)(16)(17)(18)(19) supports binding of specific antigens in those cases.
Finally, because more cases using unmutated Vκ genes lie upstream on the Vκ locus ( Figure S1), receptor editing may have taken place in these cells (45,46). Because unmutated B-CLL cases are enriched in poly/autoreactivity (47), receptor reconfiguration in these cases may not have been effective in eliminating low-affinity autoreactivity (48), due to the use of certain germline V L (49) and V H (50,51) genes and rearranged HCDR3 segments (52). This phenomenon has been observed in transgenic murine models (53,54). Additional studies that compare IgV mutation status with antigen binding will be necessary to confirm this possibility.