Multiple productive immunoglobulin heavy chain gene rearrangements in chronic lymphocytic leukemia are mostly derived from independent clones

Abstract

In chronic lymphocytic leukemia, usually a monoclonal disease, multiple productive immunoglobulin heavy chain gene rearrangements are identified sporadically. Prognostication of such cases based on immunoglobulin heavy variable gene mutational status can be problematic, especially if the different rearrangements have discordant mutational status. To gain insight into the possible biological mechanisms underlying the origin of the multiple rearrangements, we performed a comprehensive immunogenetic and immunophenotypic characterization of 31 cases with the multiple rearrangements identified in a cohort of 1147 patients with chronic lymphocytic leukemia. For the majority of cases (25/31), we provide evidence of the co-existence of at least two B lymphocyte clones with a chronic lymphocytic leukemia phenotype. We also identified clonal drifts in serial samples, likely driven by selection forces. More specifically, higher immunoglobulin variable gene identity to germline and longer complementarity determining region 3 were preferred in persistent or newly appearing clones, a phenomenon more pronounced in patients with stereotyped B-cell receptors. Finally, we report that other factors, such as TP53 gene defects and therapy administration, influence clonal selection. Our findings are relevant to clonal evolution in the context of antigen stimulation and transition of monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia.

Introduction

Chronic lymphocytic leukemia (CLL) is characterized by the expression of restricted sets of B-cell receptors (BcR), often with highly similar, stereotyped antigen-binding sites,1,2 strongly indicating the role of antigen in CLL development.3–5 Further evidence in support of this notion has been amply provided by the fact that the mutational status of the clonotypic immunoglobulin heavy variable gene (IGHV) stratifies CLL patients into two groups with markedly different prognoses.6,7 This implies that signals conveyed through BcR with distinct molecular structure likely affect the biological behavior of the CLL clone,8,9 thus contributing to the eventual clinical outcome.

A subgroup of CLL cases carry multiple (mostly double) productive IGH rearrangements (MP-IGH), and pose an exception to the rule ‘single clone – single rearrangement’. Such cases have been reported repeatedly and independently by several groups, and they are estimated to account for approximately 2% of all cases of CLL.10 The true biological and clinical implications are currently unknown, especially when one of the rearrangements is mutated while the other unmutated.

Several mechanisms have been previously linked to the phenomenon of double productive IGH rearrangements. These mechanisms involve two main themes: lack of allelic exclusion at the IG loci11 or the presence of two clonal populations.12–16

Allelic exclusion regulates the expression of IG genes in any given B lymphocyte so that it may express a single heavy chain and a single light chain. This mechanism is critical for the process of clonal selection and the generation of high-affinity, antigen-specific antibodies. In a minority of B cells, the mechanism can be disrupted, leading to lack of allelic exclusion which is characterized by the production of two functional IGH or IGK/L molecules in a single B lymphocyte.17

In CLL, lack of allelic exclusion on heavy chains leading to the presence of double productive IGH rearrangements was first reported in 1997.11 At roughly the same time, IGHV gene replacement was suggested as another molecular mechanism that could lead to the presence of double productive IGH rearrangements in CLL.18 Whatever the precise molecular mechanism(s) underlying double IGH rearrangements in a single cell/clone, these have been interpreted as evidence for the possible operation of receptor editing in CLL. This is similar to what has been reported for normal B cells, where secondary rearrangements occurring after the expression of a potentially harmful BcR can offer the cell the opportunity to evade apoptotic death.19

Double productive IGH rearrangements can also indicate the presence of two clonal populations, each expressing distinct BcR. Several cases with coexistence of two CLL clones have been reported,12–16 challenging the prevailing notion of CLL being a monoclonal disease. On the other hand, detection of double productive IGH gene rearrangements could also represent co-existence of CLL with another B lymphoproliferative disorder.12

Clonal drift is a phenomenon in lymphoid malignancies with multiple productive antigen receptor gene rearrangements, in particular T-cell large granular lymphocyte leukemia, referring to a dynamic process of alterations in the proportion of the malignant clones.20 Clonal drift has never been examined in CLL, though potentially relevant given evidence that the proliferation and overall biological behavior of CLL cells may differ between clones with mutated or unmutated IGHV.21,22

Previous studies analyzing CLL cases with MP-IGH rearrangements lack a detailed genomic analysis of IG light chains and partial IGHD-IGHJ rearrangements that can be extremely informative about the molecular status of the IG loci, thus contributing to clarification of the mechanisms involved. In the present study, we performed a comprehensive analysis of IG heavy and light chain gene rearrangements in MP-IGH CLL patients with the aim of obtaining molecular insight into the biological causes of this phenomenon. Also, for the first time, we attempted a systematic study of clonal drift in MP-IGH CLL by tracing each rearrangement at different time-points in the natural history of the disease.

Methods

Study group

MP-IGH cases were sought among 1147 CLL patients tested for IGHV gene mutational status at the University Hospital Brno, Czech Republic, from 2003 to 2011. All patients included in this study fulfilled the International Workshop on CLL/National Cancer Institute diagnostic criteria for CLL.23 Blood samples were taken after written informed consent in accordance with the Declaration of Helsinki under protocols approved by the Ethical Committee of the University Hospital Brno.

Sample processing, nucleic acid isolation and complementary DNA synthesis

B lymphocytes were routinely separated from peripheral blood using RosetteSep kits (StemCell). The purity of enriched B cells and expression of surface markers were evaluated by flow cytometry; all separated samples contained >98% CLL cells. Genomic DNA (gDNA) was isolated using the DNeasy Blood & Tissue Kit (Qiagen). For RNA isolation, either TriReagent (MRC, Inc.) or the RNA mini Kit (Qiagen) was used. Complementary DNA (cDNA) was synthesized using SuperScript II Reverse Transcriptase (Invitrogen) from 500 ng of total cellular RNA.

Polymerase chain reaction amplification of IG gene rearrangements

IGHV-IGHD-IGHJ rearrangements were amplified by reverse transcription polymerase chain reaction (PCR) using primers specific for the leader region of IGHV genes along with a consensus primer for the IGHJ genes. In cases with multiple amplicons, the PCR for IGHV-IGHD-IGHJ rearrangements was repeated on gDNA with a combination of primers for the framework region 1 (FR1) and a consensus IGHJ primer.24

PCR amplification of partial IGHD-IGHJ rearrangements was performed on gDNA utilizing seven subgroup-specific IGHD primers in combination with a consensus IGHJ primer.24 IGKV-IGKJ and IGLV-IGLJ rearrangements were amplified with primers specific for the FR1 and the IGKJ or IGLJ genes,25,26 and/or following the BIOMED-2 protocol,24 both on cDNA and gDNA.

IG sequence analysis and interpretation

PCR amplicons were subjected to direct sequencing on both strands. If multiple IGHV-IGHD-IGHJ rearrangements were amplified from the same IGHV-specific primer, subcloning was performed following recommended strategies.10 The sequences obtained were analyzed using IMGT® and the IMGT/V-QUEST tool (http://www.imgt.org).27 For partial IGHD-IGHJ rearrangements, sequence analysis was performed by a multistep procedure using BLAST (http://blast.ncbi.nlm.nih.gov/), ExPASy (http://au.expasy.org/), and IMGT® tools.

Molecular monitoring of B-cell clones over time

For long-term molecular monitoring of clonal dynamics, allele-specific oligonucleotide assays for quantitative real-time PCR (ASO-qPCR) were designed. gDNA was used for quantification of proportions of IG rearrangements. If gDNA was not available in serial samples or the ASO-qPCR assay design was not successful, clonal dynamics was assessed semi-quantitatively based on fragment analysis from cDNA using consensus FR1 and IGHJ primers. In such cases, clone size was estimated according to the size of the area under the curve.

Further details of various aspects of the methods are available in the Online Supplementary Methods.

Results

Multiple productive IGHV-IGHD-IGHJ gene rearrangements in chronic lymphocytic leukemia: incidence and overview of IGH gene repertoires

Within a cohort of 1147 CLL patients analyzed for IGHV mutational status in this study, 548 (46.3%) sequences carried mutated and 635 (53.7%) unmutated IGHV, following the 98% identity cut-off value. The skewing to unmutated cases results from the fact that our Department is a tertiary center to which cases with a less favorable clinical course are referred.

Multiple productive IGHV-IGHD-IGHJ rearrangements (MP-IGH) were identified in 31/1147 cases (2.7%). The patients’ characteristics are presented in Online Supplementary Table S1 and Online Supplementary Figure S1. Two or three transcribed productive IGHV-IGHD-IGHJ rearrangements were found in 26 (84%) and 5 (16%) cases, respectively, resulting in a total of 67 sequences. Of these, 29/67 (43%) carried mutated IGHV, while the remainder (38/67, 57%) carried unmutated IGHV; 27/38 unmutated rearrangements had IGHV with 100% identity to germline (‘truly unmutated’3). Hence, the distribution of the sequences obtained in MP-IGH cases with regards to IGHV mutational status was similar to that of the entire cohort.

The IGHV, IGHD, and IGHJ gene repertoires in MP-IGH cases did not differ significantly from those of cases with single productive rearrangements (Online Supplementary Table S2). Notably, 15/31 (48%) of MP-IGH cases harbored at least one rearrangement with a stereotyped VH CDR3 region; three of 15 such cases carried two IGHV-IGHD-IGHJ rearrangements assigned to different subsets. From the perspective of individual sequences, 18/67 (26%) of rearrangements from MP-IGH cases were stereotyped.

We compared the IG gene repertoire, somatic hypermutation status and CDR3 features between co-existing rearrangements within each case. Altogether, 41 pairs of rearrangements were analyzed; in the five cases with three rearrangements, all three possible pairs were included in the analysis. The main finding concerning IGHV usage was that the predominant pairings were IGHV3+IGHV4 and IGHV1+IGHV3 (8/41 pairs each) whereas the frequency of IGHV3+IGHV3 was low (2/41 pairs). With regards to somatic hypermutation, concordant IGHV mutational status was seen in 21/31 cases (67.7%), of which nine (29%) carried only mutated IGHV genes while the remaining 12 (38.7%) carried only unmutated IGHV genes. Ten of 31 cases (32.3%) were discordant for somatic hypermutation since they carried rearrangements of different mutational status (according to the 98% cut-off value). Detailed results of this assessment are listed in Online Supplementary Table S3.

Figure 1.Immunophenotypig of MP-IGH CLL cases. In Patient 1132, as an example of typical CLL immunophenotype, both malignant clones differed only in light chain isotype expression. Patient 862 displayed the typical immunophenotype of monoclonal CLL with IGκ light chain expression. Nevertheless, changes in proportion of the two identified IGH rearrangements were observed (see Figure 2). In Patient 1037, the malignant populations displayed distinct immunophenotypic features implying the co-existence of CLL/SLL with atypical CLL or other lymphoproliferative disorder. Mantle cell lymphoma was excluded in this patient because of cyclin D1 negativity, and a diagnosis of SLL was established (bulky disease, absolute B lymphocyte count under 5,000/μL). In Patient 1054, two clonal populations were identified; the possibility of another indolent lymphoproliferative disease co-existing with CLL could not be excluded (CLL Matutes score for the atypical clone: 2 – 3).

Multiple IGHV-IGHD-IGHJ gene rearrangements in chronic lymphocytic leukemia: immunophenotypic and molecular hints regarding their origin

We questioned whether MP-IGH could co-exist in a single clone, alluding to a lack of allelic exclusion, or whether they derived from multiple co-existing clonal B-cell populations. First, we performed detailed flow cytometry analysis in 22 cases (Figure 1). Twenty of the 22 cases had a homogeneous phenotypic profile suggestive of CLL. Interestingly, 7/20 cases had clear evidence of two coexisting CLL clones with different light chain restriction. In the remaining 2/22 cases, we could document the presence of a CLL population co-existing with another clonal B-cell population with a distinct immunophenotype (cases #1037 and 1054; Figure 1).

We then performed immunogenetic gDNA-based analysis in 26 MP-IGH cases. The reasoning behind this approach is that since a single cell and, by inference, a single clone carries only two IGH alleles, then the expected maximum number of IGH rearrangements per cell/clone is only two. Hence, the detection of partial IGHD-IGHJ (P-DJ) or non-transcribed/unproductive IGHV-IGHD-IGHJ rearrangements in MP-IGH CLL cases might constitute convincing molecular evidence in favor of the existence of multiple clonal B-cell populations, even when displaying a uniform immunophenotype. To exclude coincidental amplification or amplification of germline IGHD7-IGHJ1 region, all PCR products were sequenced and particular IGHD and IGHJ genes were assigned.

Overall, 20/26 cases were positive for P-DJ, indicating the existence of multiple clones. In 6/26 cases (four of them positive for P-DJ), this presumption was further supported by detection of an additional IGHV-IGHD-IGHJ rearrangement that was either non-transcribed productive (1 case), or unproductive due to an out-of-frame junction (5 cases).

We subsequently extended the immunogenetic analysis to the IG light chains for all 31 MP-IGH cases. Overall, 74 IGKV-IGKJ and IGLV-IGLJ clonal rearrangements were amplified. Twenty-one cases (68%) carried multiple light chain rearrangements; at least two rearrangements were productive in 18/21 cases (86%); multiple productive and transcribed rearrangements were detected in 16/18 cases (52% of all 31 patients).

Additionally, in 26 cases we analyzed rearrangements of the IGK loci involving the kappa-deleting element (KDE). Among kappa-expressing cases (14/26), ten had PCR evidence of KDE rearrangements. In the only lambda-expressing case, both IGKV-KDE and IGKJ-C-INTRON-KDE rearrangements were detected. All cases expressing both kappa and lambda light chains (11/26) were positive for KDE rearrangements. In total, all 26 analyzed MP-IGH cases had at least one rearranged IGK allele.

When combining immunophenotypic and molecular results, we could categorize the 31 MP-IGH cases into three groups (Table 1, Online Supplementary Table S4, and Online Supplementary Figure S2). Group I, defined by definite co-existence of two clonal B-cell populations, comprised nine cases (29% of MP-IGH cases; 0.79% of the whole CLL cohort), of which seven concerned co-existing CLL populations with different light chain restriction, while the remaining two concerned coexisting CLL + other B-cell clone (cases #1037 and #1054). Group II, defined by highly likely co-existence of at least two clonal populations with CLL-like phenotype, consisted of 16 cases (52% of MP-IGH cases; 1.40% of the whole CLL cohort). Group III, consisting of cases indeterminate as to one or more CLL-like populations, was formed of six cases (19% of MP-IGH cases; 0.52% of the whole CLL cohort) in which we failed to obtain conclusive evidence of more than one clone.

Table 1.Immunogenetic and immunophenotypic data and resulting category referring to the possibility of co-existence of multiple clones in the respective patient.

Figure 2.Examples of evolution of biclonal/triclonal CLL. Changes in the relative proportions of the malignant clones assessed using ASO-qPCR assays and flow cytometry were usually accompanied by alterations in absolute count of clonal B lymphocytes (the additional axis “B cell count”) and accelerated after therapy and/or by co-selection of unfavorable genomic aberrations, such as a TP53 defect. Time axes are scaled in month/year. Patient 604: at diagnosis, three rearrangements – two with unmutated IGHV1–69 (subset 5 with 20 amino acids in VH CDR3; non-stereotyped with VH CDR3 of 24 amino acids) and one with mutated IGHV2-5 gene (in gray) – were assessed. The IGHV2-5 gene rearrangement was minor and diminished during the disease course. Both IGHV1-69 gene rearrangements have persisted over time. The patient did not receive any therapy during the follow-up. Patient 862: at diagnosis, two IGHV-IGHD-IGHJ gene rearrangements utilizing unmutated IGHV1-2 and IGHV1-69 genes were detected. The IGHV1-69 gene rearrangement was diminishing during the disease course. The patient did not receive any therapy during the follow-up. Patient 261: originally biclonal disease, both clones harbored unmutated IGHV1-69 gene rearrangements (subset 7 with VH CDR3 of 23 amino acids; non-stereotyped with 29 amino acids in VH CDR3). The proportions of the clones changed, especially with therapy administration. After second-line therapy, one clone was diminishing and, at the same time, de novo TP53 mutation/deletion was detected in the second persisting clone with stereotyped BCR. Patient 279: originally monoclonal CLL (unmutated IGHV3-21, subset 2); the second CLL clone harboring unmutated IGHV1-69 (subset 3) and a complex karyotype (including TP53 defect) appeared in relapse of the disease after the first therapy. Selection of the new malignant clone led to fulminant progression of the disease, and the patient died of infectious complications (†). mut: mutated TP53 gene; wt: wild-type TP53 gene, F: fludarabine, C: cyclophosphamide, R: rituximab, A: alemtuzumab.

Tumor dynamics: molecular monitoring over time suggesting clonal drift

In 22/31 MP-IGH patients (71%), reverse transcription PCR analysis of IGHV-IGHD-IGHJ rearrangements was performed repeatedly at several time points during the course of the disease (median number of tests, 2.5; range, 2–6). The median interval from the first to the last analysis was 24 months (range, 8–74 months). Additionally, in 11 of the 22 cases with available consecutive gDNA samples, ASO-qPCR assays were designed for all IGHV-IGHD-IGHJ rearrangements to verify the results of PCR analysis and to quantify changes in the relative proportions of the clonal populations over time (Figure 2; Online Supplementary Table S5). The observed changes were evaluated through categorizing the detected clonal IGHV-IGHD-IGHJ rearrangements as: (i) diminishing (tendency to decrease in successive samples, detectable → undetectable state, >10% proportion change using ASO-qPCR), (ii) persistent (present constantly, stable or expanding compared to other persistent or diminishing rearrangement, respectively), or (iii) appearing (originally undetectable, expanding tendency).

Altogether, a clonal drift represented by changes in the proportions of the rearrangements was detected in 19/22 MP-IGH cases (86%). An IGHV-IGHD-IGHJ rearrangement, absent at initial testing, appeared in 3/19 patients besides the original rearrangement(s), whereas in 18/19 patients one of initially multiple detected IGHV-IGHD-IGHJ rearrangements diminished during the disease course (Online Supplementary Table S1). Importantly, among the latter cases, diminishing of the IGHV-mutated clone with concurrent persistence of the co-existing IGHV-unmutated clone resulted in re-categorization of seven of ten patients (70%) with originally discordant mutational status to the group with unmutated IGHV genes (Table 2).

Considering the clinical course of the 19 patients with clonal drift, we noted that the changes in the proportions of clonal populations were frequently related to progressive lymphocytosis or overall disease progression (8 out of 13 cases with progressive disease; 62%). Furthermore, eradication of a clone was observed in relation to therapy administration when one of the clones present before therapy was not detected at disease relapse while the other clone expanded (5 of 8 treated patients; in the remaining 3 patients one clone had diminished before therapy). Altogether, clonal drift was associated with disease progression/therapy in 12 of 13 progressive cases (92%; 63% of all cases with clonal drift).

Table 2.Overview of MP-IGH CLL cases with originally discordant mutational status and the impact of clonal drift on overall IGHV mutational status (clones prevailing during the disease course are shown in bold).

Figure 3.Selection of higher IGHV gene identity and/or longer VH CDR3 in patients with clonal drift. Each spot of the plot represents the difference in IGHV gene identity and VH CDR3 length in favor of the IG gene rearrangement that predominated in an individual patient during the disease course. Specifically, the position of a spot on the x-axis was calculated as the IGHV gene identity difference of persistent minus diminishing IG gene rearrangement in any individual patient, appearing minus persistent rearrangement, or appearing minus diminishing rearrangement. The position on the y-axis was calculated for VH CDR3 length difference in a similar way. White and black spots represent selection of stereotyped and non-stereotyped BcR IG, respectively. The table lists the numbers of cases (counted per IG pairs) with similar behavior over time in relation to the preference/selection of the monitored immunogenetic features. Counts of cases with selection of a stereotyped BcR IG are shown in brackets.

Since the molecular features of IGHV-IGHD-IGHJ rearrangements attest to the role of selection by antigen in CLL development,4,5 they could also be relevant to the emergence, persistence, and drift of individual clones. Thus, in the 19 patients with clonal drift, we compared IGHV-IGHD-IGHJ rearrangements in co-existing pairs (24 in total). Significantly, this analysis was suggestive of selection for clones with: (i) higher IGHV gene identity to germline and/or (ii) longer VH CDR3 (cumulative preference in 71% of pairs, P=0.005, χ test; Figure 3).

Moreover, we observed that the IG rearrangement bearing a VH CDR3 assigned to a stereotyped subset was preferred in nine pairs (82% of the pairs with at least one stereotyped BcR; 38% of all pairs with clonal drift) and, strikingly, the aforementioned tendency towards selection for higher IGHV identity and longer VH CDR3 was particularly pronounced in this subgroup (P=0.01; χ test) (Figure 3; Online Supplementary Table S3).

Genomic background: influence of TP53 gene defects on clonal drift

Genomic abnormalities have been documented to affect the survival and time to first therapy of CLL patients,28 with defects of the TP53 gene being shown to have the strongest impact on clinical outcome.29,30 Being interested in whether such defects present in co-existing clones could influence selection of one over the other, we detected TP53 defects consecutively during the disease course in five patients among the MP-IGH cohort (16%). In these patients, TP53 mutation and/or 17p deletion and other genomic abnormalities were assigned to individual clones based on: (i) changes in consecutive cytogenetic results that were correlated with changes in the relative proportions of IG rearrangements, and (ii) multiplex ligation-dependent probe amplification and TP53 sequencing of individual FACS-sorted populations. Of significance, in all cases, the TP53 defective clone expanded to the detriment of the TP53 unaffected clone (Table 3). In two cases, the selection for the TP53 defect was therapy-related. Notably, both selected clones harbored stereotyped rearrangements of the IGHV1-69 gene (subsets 3 and 7)31 (Figure 2).

Table 3.Comparison of individual B lymphocyte clones in patients with clonal drift and a TP53 defect. In all cases, a TP53 defect was related to a distinct B lymphocyte clone whereas another clone harbored intact TP53 genes.

Discussion

CLL is usually a monoclonal disease, hence, the entire CLL population constituting the progeny of a single B lymphocyte can be characterized by a unique IGHV-IGHD-IGHJ rearrangement. Significantly, stratification of patients into groups with distinct prognoses is possible through determination of IGHV mutational status.32 In MP-IGH CLL cases, assignment to the IGHV-mutated or unmutated category can be difficult, especially when the rearrangements have discordant mutational status, precluding conclusive interpretation.10 In line with our observations, the incidence of MP-IGH reaches approximately 2% of CLL cases.10

We attempted the first systematic assessment of MP-IGH CLL by performing detailed immunophenotypic and molecular profiling with the aim of elucidating the biological cause. Based on the evidence for co-existence of multiple B-cell clones, we assigned these cases into three groups. Group I (29% of MP-IGH cases; 0.79% of the whole CLL cohort) included cases for which we were able to confirm biclonal expansions differing in light chain restriction. The majority displayed typical CLL immunophenotype for both clonal populations. In two cases, the CLL clone co-existed with another B-cell clone expressing an immunophenotype atypical for CLL. Sanchez and colleagues12 previously reported a higher incidence of CLL cases with two or more phenotypically distinct B-cell clones (~4% when considering typical and atypical CLL together) than was observed in our study. This discrepancy is most likely caused by differences in methodological design. In particular, Sanchez et al. investigated the incidence of more B-cell clones in a cohort of patients with leukemic chronic lymphoproliferative disorders characterized by detailed flow cytometry,12 whereas we undertook a comprehensive molecular immunogenetic profiling complemented by fluorescence in situ hybridization (FISH) and flow cytometry studies. Furthermore, we followed a stringent approach for assigning patients to group I, requiring different light chain restriction, because alterations in the expression of other markers could be linked to intraclonal diversification or clonal evolution.

Group II (52% of MP-IGH cases; 1.40% of the whole CLL cohort) consisted of cases with immunogenetic evidence of more than one B-cell clonal population based on the number of detected IG rearrangements exceeding the allele capacity of a single cell/clone, yet in which only one homogeneous population was assessed by flow cytometry. This probably reflected either a very similar immunophenotype of the clones with the same light chain restriction, or a low proportion of one clone in the sample, or both. We were able to document this presumption using ASO-qPCR in five cases. In addition, changes in the relative proportions of the IGH rearrangements were observed over time.

For group III (19% of MP-IGH cases; 0.52% of the whole CLL cohort), we did not obtain definitive evidence mainly due to the lack of available material. Potential explanations could still relate to molecular mechanisms such as BcR editing through IGHV gene replacement18 or lack of allelic exclusion11 leading to the expression of multiple IGHV-IGHD-IGHJ rearrangements in a single cell. The first possibility (i.e. IGHV gene replacement) was effectively ruled out since we did not identify common VH CDR3 motifs co-existing in any patient.33

Although the evidence from cases in groups I and II suggests the presence of multiple B-cell clones, alternative options must be considered, including: (i) the presence of extra copies of the IGH locus due to trisomy 14 or amplification of 14q; nonetheless, this possibility is not supported in any case with available cytogenetic data (FISH and/or metaphase cytogenetics in 84% of MP-IGH cases); and, (ii) as already mentioned for group III, lack of allelic exclusion leading to dual IGH-expressing B cells, as reported for autoreactive B cells17 as well as indirectly for CLL.11,18 It is worth mentioning that in group II, we identified one case (#974) with dual expression of surface Igκ and Igλ light chains as a possible consequence of receptor editing or allelic inclusion in light chain loci.34 So far, we have not been able to document whether both IG heavy transcripts detected in this patient were also translated and expressed. Admittedly, however, only analysis at the single cell level could reliably identify the underlying mechanism(s) in the above mentioned case and also confirm the presumption made for the whole group II.

Monoclonal B-cell lymphocytosis (MBL) is regarded as a pre-malignant state of CLL.35 In contrast to the monoclonal nature of CLL, two or more “low-count” co-existing MBL clones have been documented at a high frequency.36 Moreover, persistent as well as transient MBL clones have been observed.37 Thus, CLL cases with MP-IGH gene rearrangements might represent the co-existence of CLL with a CLL-like MBL population, at least for a subset of cases. Following this line of reasoning, our cohort of MP-IGH CLL also featured a number of low-count clones (see group II above), which may signify borderline MBL/CLL clones co-existing for a certain period with the CLL clones eventually prevailing. This hypothesis is also supported by our experience with a case carrying two mutated IGHV-IGHD-IGHJ rearrangements that was originally classified as clinical MBL and, thus, excluded from the cohort studied herein. CLL eventually developed with only one of the original two rearrangements identified at the CLL stage.

Moreover, the idea of multiple B-lymphocyte clones initiating CLL is in the line with the process of antigen stimulation generally considered to contribute to the development of CLL.3–5,38 Initially, several B lymphocytes with different BcR specificities could target different epitopes of the same (auto)antigen. Later on, only some of these clones eventually gain additional abnormalities driving clonal expansion and profit from favorable interactions within their microenvironment.39 Thus, although only one clone prevails in the majority of CLL cases, many B lymphocyte clones could be expanded at the beginning. Our present results support this notion since multiple rearrangements were often detected in early stages of the disease at diagnosis (see Online Supplementary Figure S1 showing a comparison between the MP-IGH cases and cases with only a single productive IGH rearrangement).

Clonal drift as a dynamic process of altering proportions of malignant lymphocyte clones is highly relevant to the understanding of the co-existence of multiple clones and the eventual prevalence of one over the other. It was first described in T-cell large granular lymphocyte leukemia harboring two T-cell receptor beta chain gene rearrangements.20 Clonal drift had not been referenced in CLL until now. Indeed, a previously published study delineated a relative stability of neoplastic clones.12 Based on our data, it seems that the emergence of an additional clone is possible but less likely. Similarly, the disappearance of a clone seems to occur more frequently if it coexists with a more aggressive one. Our results show that higher IGHV gene identity and/or longer VH CDR3 regions were preferred over time, a phenomenon more pronounced in patients with stereotyped BcR. Relevant to these observations, telomere length measurements21 have shown that proliferation of leukemic cells with unmutated IGHV is more intense compared to that of cells with mutated IGHV. Furthermore, it is worth mentioning that longer VH CDR3 are a frequent feature of auto- and multi-reactive cells,40,41 which is also in line with the reactivity profile of IGHV-unmutated CLL clones. This indicates that antigen drive may underline clonal drift leading to selection for more aggressive clones with distinctive molecular features. Overall, based on the results of our study, clonal drift, predominantly leading to shrinkage or disappearance of clones detected at diagnosis, could be one of the factors contributing to the differing proportions of IGHV-mutated and IGHV-unmutated cases in MBL versus CLL.35,42

From a different perspective, it is reasonable to presume that genomic abnormalities might also have an impact on clonal drift. We document that the presence of a TP53 defect in individual clones can also be implicated in clonal drift, further supporting the aggressiveness of p53-defective clones, which may underline selection leading to clonal predominance29 and monoclonal CLL. In such cases, the p53 defect might represent a stronger phenotype prevailing over recognized unfavorable immunogenetic features. The impact of other genomic abnormalities, such as the 11q deletion, another strong marker of clinical outcome, remains to be elucidated given the low number of cases studied with the respective defects.

Finally, clonal drift might have important implications for decisions related to stratification and clinical management of MP-IGH CLL, especially in cases with discordant IGHV mutational status which are inconclusive regarding prognosis.10 We observed a shift in favor of IGHV-unmutated status in the majority of cases with a discordant status. This might be an explanation for recently published data indicating that patients with discordant status have an adverse clinical course similar to that of IGHV-unmutated cases.43 Moreover, based on our observations, the gradual prevailing of one clone over other, also in cases with concordant status, was often accompanied by disease progression. We, therefore, advocate repeated testing of IGHV mutational status in MP-IGH CLL since it can indicated changes in disease behavior.

In conclusion, our results suggest that most CLL cases with multiple IGHV-IGHD-IGHJ rearrangements can be accounted for by the presence of multiple B lymphocyte clones with CLL or CLL-like phenotype co-existing within the same patient. Importantly, we document for the first time that their proportions may change over time and that these changes are likely influenced by the molecular features of the BcR, including IGHV mutational status and VH CDR3 composition and length, and by the genomic make-up of the clones as well. This is potentially critical for understanding what drives B lymphocyte clonal evolution and could, therefore, provide insights and the means to influence or even halt this process.

Acknowledgments

The authors thank local clinicians from Boskovice, Breclav, Kromeriz, Trebic and Znojmo for their cooperation in collection of the samples and patients’ data, the patients who were willing to participate in the study, Cyril Handlir and Mikulas Stribrny for helpful advice and stimulating discussions, and Jana Kotaskova for help with preparation of a cohort of control patients. The authors also thank the Czech Leukemia Study Group for Life (CELL) and the European Research Initiative on CLL (ERIC) for support.

Footnotes

• The online version of this article has a Supplementary Appendix.
• Funding This project was supported by grants IGA MZ CR NT13493-4/2012 and NS10439-3/2009; MSMT projects CZ.1.05/1.1.00/02.0068 (CEITEC) and CZ.1.07/2.3.00/20.0045 (OPVK SuPReMMe); MUNI/A/0723/2012 and project MHCZ-DRO FNBr65269705.
• Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.

• Received March 7, 2013.
• Accepted September 9, 2013.