Chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis (MBL) with (MBLhi) or without (MBLlo) absolute B-lymphocytosis precedes most CLL cases,the specific determinants for malignant progression remaining unknown.
For this purpose, simultaneous iFISH and molecular analysis of well-established cytogenetic alterations of chromosomes 11, 12, 13, 14 and 17 together with the pattern of rearrangement of the IGHV genes were performed in CLL-like cells from MBL and CLL cases. Our results based on 78 CLL-like MBL and 117 CLL clones from 166 subjects living in the same geographical area, show the existence of three major groups of clones with distinct but partially overlapping patterns of IGHV gene usage, IGHV mutational status and cytogenetic alterations. These included a group enriched in MBLlo clones expressing specific IGHV subgroups (e.g. VH3-23) with no or isolated good-prognosis cytogenetic alterations, a second group which mainly consisted of clinical MBLhi and advanced stage CLL with a skewed but different CLL-associated IGHV gene repertoire (e.g. VH1-69), frequently associated with complex karyotypes and poor-prognosis cytogenetic alterations, and a third group of clones with intermediate features, with prevalence of mutated IGHV genes, and higher numbers of del(13q)+ clonal B-cells.
These findings suggest that the specific IGHV repertoire and IGHV mutational status of CLL-like B-cell clones may modulate the type of cytogenetic alterations acquired, their rate of acquisition and/or potentially also their clinical consequences. Further long-term follow-up studies investigating the IGHV gene repertoire of MBLlo clones in distinct geographic areas and microenvironments are required to confirm our findings and shed light on the potential role of some antigen-binding BCR specificities contributing to clonal evolution.
Citation: Henriques A, Rodríguez-Caballero A, Nieto WG, Langerak AW, Criado I, Lécrevisse Q, et al. (2013) Combined Patterns of IGHV Repertoire and Cytogenetic/Molecular Alterations in Monoclonal B Lymphocytosis versus Chronic Lymphocytic Leukemia. PLoS ONE 8(7): e67751. doi:10.1371/journal.pone.0067751
Editor: Paul G. Thomas, St. Jude Children’s Research Hospital, United States of America
Received: January 23, 2013; Accepted: May 21, 2013; Published: July 3, 2013
Copyright: © 2013 Henriques et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: AH was supported by a grant from the Fundação para a Ciência e Tecnologia of Portugal (SFRH/BD/31609/2006), ARC was supported by a grant from Fundación Científica de la Asociación Española contra el Cáncer (AECC-2008). This work was financially supported by the following grants: Red Temática de Investigación Cooperativa en Cáncer del Instituto de Salud Carlos III - FONDOS FEDER (RD06/0020/0035); FIS PI06/0824-FEDER, PS09/02430-FEDER and FIS PI12/00905, from the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain; GRS206/A/08 from the Gerencia Regional de Salud de Castilla y León and Ayuda al Grupo GR37 de Excelencia de Castilla y León, Consejería de Educación; SAN/1778/2009, Consejería de Sanidad, Junta de Castilla y León, Valladolid, Spain and FS/1-2010 Fundación Memoria D. Samuel Solórzano, Universidad de Salamanca, Salamanca, Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Monoclonal B lymphocytosis (MBL) is defined by the presence of a low to moderate expansion of circulating clonal B lymphocytes (<5×109/L) –most frequently resembling the phenotype of chronic lymphocytic leukemia (CLL) cells (CLL-like cells)– in otherwise healthy adults, in the absence of symptoms and signs of an underlying chronic lymphoid malignancy , . Recent multiparameter flow cytometry studies have demonstrated that CLL-like MBL clones can be found in a significant proportion of healthy adults over 40 years. Their frequency ranges from 3.5% to around 12% of the general population, and between 13% to 18% of first-degree relatives of CLL patients, depending on the sensitivity of the technique . Although in most CLL-like MBL cases, MBL clones are associated with a stable and indolent clinical course, a small proportion of cases presenting with lymphocytosis will eventually progress to CLL , . On the other hand, it has been shown that virtually every CLL is preceded by an MBL, which may have remained stable for variable periods of time . Identification and full characterization of the phenotypic and genetic features of CLL-like MBL cells in the absence (MBLlo) and presence (MBLhi) of an absolute B-lymphocytosis, versus CLL cells, may provide insight into the key mechanisms and events involved in the expansion of the MBL clones and their transformation to CLL, thereby contributing to a better understanding of the natural history of the disease.
Previous studies have shown that MBLhi clones may display the typical spectrum of chromosomal alterations observed in CLL, e.g. del(13q), trisomy 12, del(11q) and even del(17p); conversely, MBLlo B-cells appear to more frequently carry normal karyotypes and to a lesser extent, isolated del(13q14.3) or trisomy 12, in the absence of chromosomal alterations associated to poor prognosis CLL, such as del(17p13) and del(11q22) . These observations suggest that MBLlo, MBLhi and CLL clones could be different stages in the spectrum from reactive MBL B-cells to CLL requiring therapy. Despite this, analysis of the pattern of usage of the immunoglobulin heavy chain variable region (IGHV) gene in both MBLhi and CLL cases showed that it is not random. Accordingly, a predominant usage of specific IGHV subgroups has been reported for both MBLhi clones and mutated CLL cells (e.g. the IGHV3-23 and IGHV4-34) as well as for unmutated CLL (e.g.IGHV1-69) . In turn, very preliminary studies  indicate that MBLlo clones rarely use the IGHV4-34 subgroup, while they may display a higher frequency of IGHV4-59/61 B-cell receptor (BCR) genes, which are rarely used in CLL .
Here, we investigated for the first time the potential existence of unique cytogenetic profiles associated with specific IGHV repertoires that could be associated with an increased risk of progression from MBLlo to MBLhi and CLL. Our results, based on a series of 78 MBL and 117 CLL clones from a total of 166 subjects from the same geographical area, suggest the existence of distinct but partially overlapping molecular and cytogenetic profiles among MBLlo, MBLhi and CLL cases.
Materials and Methods
Patients and Samples
A total of 166 subjects presenting one or more CLL-like MBL and/or CLL clonal B-cell populations were included in this study: 15 cases (9%) corresponded to healthy individuals with MBLlo – <200 clonal B-cells/µL in peripheral blood (PB); 5 males and 10 females; mean age of 68±13 years; range: 49–84 years, –33 (20%) were MBLhi – ≥200 and <5,000 clonal CLL-like B-cells/µL of PB – (20 males and 13 females; mean age of 72±12 years; range: 37–89 years), –114 (69%) had newly-diagnosed untreated CLL (66 males and 48 females; mean age of 70±13 years; range: 35–89 years) and 4 (2%) had other B-cell lymphoproliferative disorders (B-CLPD) with coexistence of one or two CLL-like MBL B-cell population(s). From the 33 MBLhi cases, 20 (61%) showed clinical MBL (>2,000 clonal B-cells/µl of PB). Individuals corresponded to consecutive MBL and CLL subjects from Salamanca (Spain) and Coimbra (Portugal) in the western area of the Iberian Peninsula.
PB samples were obtained from each subject after written informed consent was given, and the study was approved by the local ethics committees of the two participating centres (University Hospital of Salamanca and Histocompatibility Centre of Coimbra). Diagnosis of MBL and CLL was based on the World Health Organization (WHO) 2008 criteria . Clinical staging of CLL subjects according to Binet classification  was collected retrospectively; 46/77 (60%) CLL cases were diagnosed as stage A and the remaining cases (31/77, 40%) as stage B/C. Overall, 37/166 subjects (22%) showed co-existence of two or three phenotypically different aberrant B-cell populations (multiclonal cases; 25 males and 12 females with a mean age of 76±8 years; range: 57–89 years), while the remaining 129 individuals showed one single phenotypically aberrant monoclonal B-cell population (monoclonal cases; 69 males and 60 females with a mean age of 68±12 years; range: 35–89 years). In 26/37 multiclonal cases, all different B-cell populations showed a typical CLL-like phenotype, while in the remaining 11 cases only one B-cell population displayed a typical CLL-like phenotype co-existing with population(s) phenotypically compatible with other B-CLPD . For this study, analysis was focused only on those aberrant B-cell populations displaying a typical CLL-like and CLL phenotype (n = 195 B-cell clones). The distribution of all CLL-like and CLL clonal populations analyzed in the distinct diagnostic categories was as follows: 27 corresponded to CLL-like MBLlo, 51 to CLL-like MBLhi and 117 to CLL (Table 1).
Immunophenotypic studies were performed on erythrocyte-lysed whole PB samples according to procedures previously described in detail . PB white blood cells (WBC) were systematically stained with the monoclonal antibody (MAb) combinations detailed in Table S1. All cases showed a clonal (imbalanced SmIgκ:SmIgλ ratio of >3∶1 or <1∶3) and/or an aberrant CD5+CLL-like B-cell population, as reported elsewhere  (see Materials and Methods S1).
Data acquisition was performed in two steps on a FACSCanto II flow cytometer – Becton-Dickinson Biosciences –(BD, San José, CA, USA) using the FACSDiva software (V6.1; BD): first, information about 1×105 events corresponding to the whole sample cellularity was stored; in the second step, information was stored exclusively for CD19+ and/or CD20+gated events, contained in a minimum of 5×106 leucocytes/tube. Instrument setup, calibration and daily monitoring were performed according to well-established protocols  (see Materials and Methods S1). For data analysis, the Infinicyt™ software (Cytognos SL, Salamanca, Spain), was used. The minimum number of clustered events required to define a B-cell population was of 50 cells.
Fluorescence-activated Cell Sorting (FACS) Purification of B-cell Populations (FACSorting)
For all individuals studied, each CLL-like CD5+ B-cell population identified was purified in a FACSAria III flow cytometer (BD). In those samples with more than one aberrant B-cell population (n = 37), discrimination among them was based on their distinct patterns of expression for ≥1 of the B-cell markers analyzed, as described elsewhere . The clonal nature of each FACS-purified B-cell population (purity: 98%±0.8%) was assessed by both cytogenetic and molecular techniques, as described below.
Cytogenetic and Molecular Studies
The presence of the most common cytogenetic abnormalities associated with CLL was investigated by multicolour interphase fluorescence in situ hybridization (iFISH) on slides containing FACS-purified and fixed aberrant B-cells, as previously described  (see Materials and Methods S1 for further details).
Analysis of the patterns of rearrangement of the IGHV genes was performed for each FACS-purified CLL and CLL-like B-cell population. Genomic DNA preparation, PCR amplification, sequencing and analysis of V, (D), J genes were performed following well-established protocols ,  (see Materials and Methods S1 for more detailed information).
Forward and reverse sequences were aligned into a single resolved sequence and then aligned with germline sequences using the IMGT database and tools (http://imgt.cines.fr). For MBLlo clones, whole genomic amplification (WGA) was performed prior to the analysis, using the RepligR UltraFast Mini kit (Qiagen, Valencia, CA) as per the recommendations of the manufacturer. For each clonal B-cell population, only in-frame rearrangements were evaluated. Sequences containing >2% deviation from the germline sequence were considered as being somatically mutated. Those MBLlo cases showing more than one productive rearrangement corresponding to different IGHV genes within each purified CLL-like B-cell population were excluded from this study, because in such cases we could not establish the precise association between each IGHV gene and the underlying cytogenetic alterations detected.
Analysis of CLL-associated NOTCH1 mutations  was performed via PCR of previously amplified genomic DNA from each FACS-purified CLL-like B-cell population for a total of 70 clones (5 MBLlo, 14 MBLhi and 51 CLL clones).
Conventional descriptive and comparative statistics –the nonparametric Kruskal-Wallis and Mann-Whitney U tests (for continuous variables), or the Pearson’s χ2 and Fisher exact tests (for categorical variables)–were performed using the SPSS software program (SPSS 15.0 Inc. Chicago, IL). P values <0.05 were considered to be associated with statistical significance.
For multivariate comparisons among MBLlo, MBLhi and CLL clones, based on the count of clonal B cells/µL and the percentage of aberrant/clonal cells carrying the different cytogenetic profiles, principal component analysis (PCA) was applied, and graphically visualized with the 3D Automated Population Separator (APS) view – Principal Component 1 (PC1) vs PC2 vs PC3– of the Infinicyt™ software (Cytognos SL, Salamanca, Spain). As previously described in detail, in this APS view, each axis of a plot is represented by a different PC as a linear combination of parameters with distinct statistical weights .
For the assignment of MBLlo, MBLhi and CLL clones to different groups, the size of the clone and the percentage of altered cells for each cytogenetic abnormality were the continuous variables included in the PCA-based assay performed with the Infinicyt software™, while IGHV gene usage, IGHV mutational status and clinical staging of CLL subjects according to the Binet classification were treated as categorical variables, used only for labelling the different clones within each group, after applying the PCA.
Overall Size and BCR Features of CLL-like MBL and CLL B-cell Clones
The median relative percentage and absolute count of CLL-like and CLL B-cells progressively increased from MBLlo (0.6% and 20 cells/µl), to MBLhi (14% and 2,000 cells/µl) and CLL clones (57% and 17,400 cells/µl) (P<0.0001) (Table 2).
Of note, around half of all MBLlo and MBLhi cell populations (52% and 51%, respectively) derived from multiclonal cases, whereas only 22% of CLL clones were identified in multiclonal cases (P≤0.03; Table 2). In addition, CLL clones less frequently showed mutated IGHV genes (53%) compared to both MBLhi (73%) and MBLlo (67%) clones (P≤0.02) (Table 2).
Cytogenetic Features and NOTCH1 Mutation in CLL-like MBL and CLL B-cell Clones
Overall, MBLlo B-cell clones showed a significantly lower frequency of genetic alterations associated with CLL (33%) than MBLhi (51%) and CLL (62%) B-cells (P≤0.02) (Table 3). Furthermore, only a small proportion of MBLlo (7%) and MBLhi clones (14%) showed coexistence of ≥2 cytogenetic alterations, while this was found in 33% of all CLL clones (P≤0.04).
Regarding each specific cytogenetic/molecular alteration, a tendency towards a greater frequency of del(13q) and trisomy 12 was observed from MBLlo to MBLhi and CLL clones, although differences only reached statistical significance for the frequency of del(13q14) involving the RB1 gene (P = 0.04). In addition, presence of t(14q32) and del(11q22.3) were exclusively found among MBLhi and CLL, while del(17p), del(11q23) and NOTCH1 mutations were only present in CLL clones (Table 3). Of note, among the cytogenetically altered clones, no significant differences were observed in the percentage of altered cells, except for a greater proportion of B-cells with trisomy 12 among both MBLhi and CLL vs MBLlo B-cell clones (P≤0.04) (Table 3).
NOTCH1 mutations occurred in 5/52 CLL cases (10%), in which a preferential association with IGHV unmutated clones (80%, P = 0.02) and a high frequency of cases (3/5, 60%) harbouring trisomy 12 as an additional isolated chromosomal abnormality (P = 0.007) was observed; in the remaining two CLL cases, the presence of NOTCH1 mutation was associated with del(13q14) involving the RB1 gene and to both del(13q14.3) and del(17p), respectively. None of the 19 MBL cases investigated showed NOTCH1 mutations.
Molecular Characteristics of CLL-like MBL and CLL B-cell Clones
Analysis of the IGHV gene revealed single in frame gene rearrangements for each clonal B-cell population analyzed. Interestingly, shorter CDR3 sequences of the IGHV gene were found among MBLlo versus CLL clones. So, the frequency of CLL clones with CDR3 sequences coding for >20 aminoacids became significantly higher than that observed among MBLlo and MBLhi clones (P = 0.02) (Table 4).
Regarding IGHV, DH and JH gene usage, no significant differences were found between the three groups of CLL-like B-cell clones, except for the VH3 and DH3 IGHV genes (Table 4): CLL showed lower frequency of VH3 usage and a greater proportion of DH3+clones vs MBLlo and MBLhi (P≤0.04) (Table 4). Of note, a significant percentage of all CLL (72%), MBLhi (74%) and MBLlo (85%) clones corresponded to only 12 IGHV genes, namely V3-23, V3-11, V5-51, V3-21, V1-2, V1-3, V4-39, V3-7, V3-30, V4-34, V3-48 and V1-69 (Figure 1). Among these, preferential usage (P<0.04) of the VH3-23 gene was observed in both MBLlo (7/27, 26%) and MBLhi (10/51, 20%) vs CLL (5/113, 4%) clones.
The three diagnostic categories studied are depicted by different colors (green, MBLlo; red, MBLhi; blue, CLL B-cell clones) and the absence vs presence of one vs≥2 chromosomal alterationsperclone, is indicated by empty circles, light colored and dark colored circles, respectively. For each IGHV subgroup, the clones are represented in the Y-axis according to the absolute number of clonal B cells per µL of PB (A) and the percentage of cells genetically altered, by iFISH (B). Different FISH patterns are defined by the following symbols in panel B:, del(13q14.3);, biallelic del(13q14.3);, del(13q14);, trisomy 12; Δ, del(11q);▿,del(17p) and; □, t(14q32); dotted contour lines in panel A highlight those clones phenotypically classified as SLL(small lymphocytic lymphoma); dotted blue lines in panel B indicate cells from the same B-cell clone showing different cytogenetic abnormalities; U = unmutated clones; a = clones with NOTCH1 mutation.
Relationship between the Most Frequently used IGHV Genes and the Cytogenetic Profile of CLL-like MBL and CLL B-cell Clones
As mentioned above, preferential usage of the VH3-23 gene was observed in both MBLlo and MBLhi versus CLL clones (Figure 1A). VH3-23+ MBL clones typically showed no cytogenetic alterations (8/17) or they carried an isolated cytogenetic alteration which corresponded either to trisomy 12 (3/17) or deletion of 13q (3/17) (Figure 1B). Nevertheless, two MBLhi clones showed co-existence of trisomy 12 and del(11q22.3) and one MBLlo clone showed del(13q) including both the 13q14.3 and 13q14 (RB1) chromosomal regions (Figure 1B). From the fiveVH3-23+ CLL clones only one carried genetic alterations –trisomy 12 and del(13q) –. Most interestingly, VH3-23+MBLlo clones frequently showed unmutated IGHV genes, including most unmutated MBLlo clones, with <10 CLL-like cells/µl(5/8; 63%), which contrasts to the much lower frequency of unmutated VH3-23 CLL clones.
A similar frequency of usage of the VH3-11, VH5-51, VH3-21 and VH1-2 genes was observed in both MBLlo and MBLhi versus CLL (Figure 1). In none of the clones expressing these IGHV genes, cytogenetic alterations associated with a poor disease outcome – e.g. del(17p) and/or del(11q) – were found; in addition, most MBL and CLL clones expressing these IGHV genes showed no cytogenetic alteration, or they just had a single abnormality. Despite this, NOTCH1 mutations were more frequently observed among cytogenetically altered, IGHV unmutated CLL clones expressing these IGHV genes (one VH3-11+, one VH3-21+ and one VH1-2+ clones). Noteworthy, 4/6 CLL cases classified as small lymphocytic lymphoma (SLL) variants were also included among cases with a VH3-11 (n = 2), VH3-21 (n = 1) and VH1-2 (n = 1) repertoire in this group.
Finally, among those clonal B-cell populations which expressed the VH1-3, VH4-39, VH3-7, VH3-30, VH4-34, VH3-48 and VH1-69 genes, CLL clones were overrepresented (61/113, 54%) versus both MBLlo (10/27, 37%) and MBLhi (18/51, 35%) clones. Notably, a high number of CLL clones carrying these IGHV genes in association with one or more cytogenetic alterations, including poor prognosis cytogenetic alterations, was found among these cases (44/61, 72%). In this regard, del(13q) including both the 13q14.3 and 13q14 (RB1) chromosomal regions was frequently detected (single or combined lesion) in these CLL and also MBLhi clones, particularly among those cases expressing the VH3-30 and VH4-34 gene genes; presence of trisomy 12, del(11q) and t(14q32) were also common among these CLL cases (16%, 13% and 18%, respectively) while being infrequent in MBL cases (only 2 MBLhi clones showed isolated trisomy 12). Moreover, del(17p) alone and complex karyotypes with ≥3 cytogenetic/molecular alterations were also found in 4 of the CLL clones which expressed the VH1-3, VH4-39, VH3-30, VH3-48, VH4-34 and VH1-69 IGH genes, respectively (Figure 1B). Remarkably, ≥1 genetic alteration was systematically detected in a major fraction of the VH1-69+ clonal cells (Figure 1A) while being absent in the few MBL clones which expressed this specific IGHV gene. Of note, unmutated IGHV genes were a hallmark of both VH1-69+ (12/14 clones; 86%) and VH4-39+ (9/10 clones; 90%), independently of their MBL vs CLL nature (Figure 1A).
Based on the observation of the above described associations, we performed a multivariate analysis based on PCA, in searching for unique patterns of association between cytogenetic alterations and IGHV repertoires among MBL vs CLL clones. Three major groups of CLL-like MBL and CLL clones were identified, according to the absolute number of clonal B cells/µL and the percentage of cells carrying cytogenetic alterations (Figure 2) and then labelled according to their pattern of IGHV gene usage and the VH mutational status. Of note, the most homogeneous group (Group 1) included virtually all MBLlo clones (77%) and half of the MBLhi clones (54%), but only around one fourth of Binet stage B/C CLL (28%); by contrast, no MBLlo clones were included in Group 3 (Figure 2D). Group 2 showed a more heterogeneous distribution with an intermediate pattern. In detail, Group 1 was mainly characterized by cases with a normal karyotype (83%) and lower numbers of cytogenetically altered cells mostly displaying the VH1-2, VH3-23 and VH4-34 IGHV genes (Table S2); in turn, Group 2 typically showed a higher number of cases with mutated IGHV genes, and higher numbers of del(13q)+ clonal B cells, while Group 3 included high numbers of cases with unmutated IGHV genes, trisomy 12 and an IGHV repertoire enriched in unmutated VH1-69+ CLL clones (Table S2).
Overall, MBLlo, MBLhi and CLL cases are clustered into groups distinguished by different colors in A: magenta, gray, and black circles (A). The distribution of MBLlo, MBLhi, CLL-stage A and CLL-stage B/C clones are coloured differently in B: MBLlo, green; MBLhi, red, CLL stage A and B/C light blue and dark blue, respectively (B). The most informative parameters contributing to the best discrimination between 1×1 comparisons of the three groups are displayed in a decreasing order of percentage contribution to each of the principal component (C); Distribution of MBLlo, MBLhi and CLL clones among the three major groups defined in panel A by principal component analysis (D); CLL, chronic lymphocytic leukemia; MBL, monoclonal B lymphocytosis; PC: principal component.
It is now well established that emergence of CLL is typically preceded by MBL . However, only a fraction of all MBLhi will evolve to CLL, at a rate of 1.1% persons/year , while the outcome of MBLlo remains unknown. Despite this, general consensus exists in that stepwise acquisition of specific genetic alterations may determine the rate of progression, not only from MBLhi to CLL, but potentially also from MBLlo to MBLhi and eventually to CLL. Concurrence of chronic antigen stimulation through specific BCRs may further support and accelerate the expansion of MBL clones, facilitate acquisition of new genetic alterations and therefore contribute to progression to CLL , . Although data has accumulated in the last decade about the cytogenetic alterations and the IGHV gene repertoire of CLL-like clonal B-cells in both MBL and CLL, to our knowledge, this is the first report about the combined patterns of cytogenetic alterations and IGHV gene repertoire in MBLlo vs MBLhi vs CLL clones.
In recent years, more than a thousand different molecular/genetic alterations reflected in multiple distinct and complex cytogenetic/molecular profiles in individual CLL patients, have been described through high-throughput whole-genome sequencing approaches , . However, only a relatively small number of cytogenetic/molecular alterations recurrently occur at relatively high frequencies (e.g. >5% cases) , . Such alterations include del(13q14), reported in around half of all CLL cases, trisomy 12, present in about one third of the patients and del(11q), del(17p), t(14q32) and NOTCH1 mutations, which occur in between 5-15% of all CLL cases , , . In around half of CLL cases, unmutated IGH genes associated with preferential usage of specific IGHV genes (i.e. VH1-69 and VH4-34) and the above described cytogenetic alterations have also been reported in CLL. In turn, MBLhi cases share molecular features with good-prognosis CLL in terms of both the IGHV gene repertoire and chromosomal alterations , , with a greater frequency of IGHV mutated cases. By contrast, preliminary data indicates that the IGHV repertoire expressed by MBLlo could be strikingly different from that of both typical CLL and MBLhi cases ; in addition, such MBLlo clones appear to display a much lower frequency of chromosomal alterations, restricted to del(13q14.3) and trisomy 12, with a high prevalence of IGHV mutated cases (similar to that of MBLhi cases) , and no poor-prognosis cytogenetic alterations , .
In line with such observations, we also found a lower frequency of both cytogenetically altered and IGHV unmutated CLL-like clones in MBLlo vs both MBLhi and CLL and vs CLL clones, respectively. Interestingly, the proportion of B-cell clones carrying ≥2 alterations significantly increased from MBLhi to CLL. On top of the progressively higher number of cytogenetic/molecular alterations found in MBLlo vs MBLhi and CLL, the cytogenetic profile of clonal B-cells also became significantly more heterogeneous among the latter two groups. Accordingly, while del(13q14.3) and to a much lesser extent, del(13q14) involving the RB1 gene and trisomy 12, were already detected in a small fraction of MBLlo clones, del(11q) and t(14q32) emerged at an MBLhi stage, whereas del(17p), del(11q23) and NOTCH1 mutations were only found in CLL. These latter three alterations typically involved CLL clones that already had other cytogenetic alterations and therefore, had more complex cytogenetic/molecular profiles. In line with these findings, the altered CLL-like MBL and CLL clones showed progressively increasing percentages of cells carrying del(13q14.3), del(13q14), trisomy 12, t(14q32),del(11q)and del(17p13.1),respectively. In accordance with previous observations , NOTCH1 mutations were exclusively detected in CLL (preferentially among unmutated CLL clones) which also had other cytogenetic alterations – e.g., trisomy 12, del(13q14) and/or del(17p).
The overall increased frequency of all cytogenetic alterations, together with the more complex cytogenetic/molecular profiles, observed from MBLlo to MBLhi and CLL would support the notion that evolution from MBLlo to MBLhi and CLL is paralleled by progressive acquisition of recurrent cytogenetic alterations, each of which appears to emerge at specific MBL and CLL stages, in line with previous data from our and other groups , . Accordingly, del(13q), and to a lesser extent trisomy 12, are relatively early cytogenetic events which may frequently occur at an MBLlo stage, whereas del(17p), NOTCH1 mutations, and to a lesser extent also del(11q) and t(14q32), would typically arise later, as secondary cytogenetic events occurring at an MBLhi or CLL stage. Acquisition of these and other genetic changes may potentially be associated with an increased proliferation and/or survival of the altered CLL-like cells. At the earliest stages of development of MBL, proliferation and/or survival signals could be provided to the MBL clone by chronically sustained BCR stimulation. If this holds true, the BCR features could also play a critical role in determining the probability of cytogenetic progression. Unfortunately, our series of MBL -particularly of MBLlo- is quite short at this time to further confirm this hypothesis, due to the difficulty in collecting cases with enough number of CLL-like B-cells, to perform in parallel reliable iFISH and molecular analyses. In this regard, the limited number of MBLlo cases included in our series may predominantly present with the genetic/molecular patterns of a low risk MBL cohort, which may not be related to CLL progression. Despite this, in accordance with other recent reports , , , non-random usage of IGHV genes with clearly different IGHV gene repertoires was found in our series in MBL vs CLL. As expected, the most frequently used IGHV genes in CLL were the VH4-34, VH3-30, VH1-69, VH3-48, VH4-39, VH1-2 and VH3-7 genes, accounting for around half of the CLL clones. Interestingly, also half of the CLL clones showed unmutated IGHV genes, strikingly high frequencies of unmutated clones being detected among cells expressing VH1-69, VH4-39 and VH1-2. By contrast, VH3-23+ B-cells predominated among the MBLlo and MBLhi clones, most VH3-23+ MBLlo cases showing very low counts of IGHV unmutated clonal B-cells. Of note, the IGHV genes used by the MBLhi clones were commonly observed in either CLL (e.g. VH4–34, VH1-2, VH3–48, and VH4–39) or MBLlo (e.g. VH3-23 and VH4-34), but usually at lower frequencies. The fact that these particular IGHV genes have been associated with previously reported stereotypic B-cell receptors in CLL clones , together with our own results which show that the complementary-determining regions (CDR3) of the IGHV genes are highly homologous in around one fifth of the B-cell clones from our short cohort (Table S3), would reinforce the role of some antigen-binding BCR specificities in clonal evolution.
Based on the overall patterns of cytogenetic alterations and IGHV gene usage together with the BCR mutational status, it could be concluded that while some unmutated IGHV genes appear to be associated with the acquisition of complex cytogenetic profiles, rapid expansion of clonal CLL-like B-cells and progression to CLL (e.g. IGHV1-69), others would not (e.g. IGHV3-23); the latter clones would show a more benign behaviour. This could potentially be due to a lower binding affinity of the unmutated BCR for the antigen, the recognition of specific subtypes of low concentrated antigens and/or unique immune response profiles. In line with this hypothesis, IGHV genes over-represented among CLL clones (e.g.VH4-39 and VH1-69) frequently corresponded to IGHV genes enriched in genes encoding for antibodies that recognise a broad variety of relatively common and abundant (auto)antigens, including low-affinity BCR, e.g. myoglobulin, thyroglobulin, actin, and ssDNA ,  associated with T-independent, type II autoimmune responses . In contrast, the unmutated IGHV3-23 BCR was over-represented among our MBLlo cases, normal peripheral blood IGHV3-23+ IgM+ B-cells being associated with recognition of superantigens –. Thus, the association between MBLlo and unmutated IGHV3-23 could be potentially due to a low affinity of this particular BCR for low concentrated/prevalent (super)antigens, which would limit the development of repetitive immune responses associated with the expansion of MBL clones and/or their cytogenetic progression. A recent study  also reported MBLlo cases to display an IGHV gene repertoire different from that of CLL patients (e.g. absence of IGHV1-69+ MBLlo clones, together with a low frequency of the IGHV4-34 gene and overrepresentation of the IGHV4-59/61 genes); however, no preferential usage of the IGHV3-23 gene was found among MBLlo cells in this series. Further studies investigating the IGHV gene repertoire of MBLlo clones in distinct geographic areas and microenvironments, may shed light on those factors accounting for such apparent discrepancies, as an association between MBLlo and previous history of infections has been recently reported in this setting .
Taken together, these results would support the notion that antigen-driven BCR-stimulation could be a triggering factor in driving CLL-like B-cells to expand, in line with recent data showing a significant association between MBL in the general population and the individual history of infectious diseases and vaccination , whilst depending on the nature of the antigenic stimuli, distinct patterns of cytogenetic changes might then occur. Thus, the specific combination of cytogenetic alterations acquired by the CLL-like B-cells may determine, for distinct antigenic stimuli, and specific BCR repertoires, the outcome of the genetically-targeted cell. Long-term longitudinal studies, ideally of the same cases at different time-points and at different stages of the disease, would be crucial to definitively confirm these hypotheses, although based on our preliminary follow-up date  this may require decades due to the stable nature of most MBLlo clones in the short-term.
In summary, MBL and CLL clones appear to display a distinct but partially overlapping pattern of IGHV gene usage, IGHV mutational status and cytogenetic alterations, which may translate into distinct groups of clones with different genetic/molecular features associated with a distinct clinical behavior. Sequential studies in larger series of cases followed for long periods of time are ongoing to investigate the risk of progression and outcome of MBL clones with specific IGHV and iFISH cytogenetic profiles.
Monoclonal antibody combinations used for the immunophenotypic analysis of CLL-like and CLL B cells.
Informative parameters of the CLL-like/CLL B-cell clones included in the three major groups graphically visualized with APS view of the Infinicyt™ software.
Heavy chain variable region (IGHV) sequences of CLL-like and CLL B-cell clones analyzed by the IMGT-V-QUEST tool.
The authors thank Maria Luz Sánchez and Paloma Bárcena for expert assistance in flow cytometry sorting of CLL-like cells, Ana Rasillo, María Laura Gutiérrez and Ana Balanzategui for expert assistance in cytogenetic/molecular studies, María Jara, Belén Espinosa and Cristina Jimenez for technical assistance, and Cristina Teodósio for expert assistance in statistical analysis. Also, the authors thank Alfonso Romero and Paulino Fernandez-Navarro for their assistance in the coordination with the Primary Health Care Group of Salamanca, as well as all members of Primary Health Care Group of Salamanca for the Study of MBL, who were directly responsible for collection of samples from the cohort of MBLlo individuals recruited.
Conceived and designed the experiments: AO JA AH ARC. Performed the experiments: AH ARC WGN IC. Analyzed the data: AH ARC AWL MG WGN JA AO. Contributed reagents/materials/analysis tools: AWL QL MLP AP. Wrote the paper: AH ARC AO JA. Provided patient samples and clinical data: MG MLP. Retrieved relevant clinical information from patient records: AP. Provided resources: AP.
- 1. Rawstron AC, Bennett FL, O’Connor SJ, Kwok M, Fenton JA, et al. (2008) Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia. N Engl J Med 359: 575–583.
- 2. Marti GE, Rawstron AC, Ghia P, Hillmen P, Houlston RS, et al. (2005) Diagnostic criteria for monoclonal B-cell lymphocytosis. Br J Haematol 130: 325–332.
- 3. Scarfo L, Dagklis A, Scielzo C, Fazi C, Ghia P (2010) CLL-like monoclonal B-cell lymphocytosis: are we all bound to have it? Semin Cancer Biol 20: 384–390.
- 4. Shanafelt TD, Kay NE, Rabe KG, Call TG, Zent CS, et al. (2009) Brief report: natural history of individuals with clinically recognized monoclonal B-cell lymphocytosis compared with patients with Rai 0 chronic lymphocytic leukemia. J Clin Oncol 27: 3959–3963.
- 5. Landgren O, Albitar M, Ma W, Abbasi F, Hayes RB, et al. (2009) B-cell clones as early markers for chronic lymphocytic leukemia. N Engl J Med 360: 659–667.
- 6. Lanasa MC, Allgood SD, Volkheimer AD, Gockerman JP, Whitesides JF, et al. (2010) Single-cell analysis reveals oligoclonality among ‘low-count’ monoclonal B-cell lymphocytosis. Leukemia 24: 133–140.
- 7. Scarfò L, Zibellini S, Tedeschi A, Maura F, Neri A, et al. (2012) Impact of B-cell count and imaging screening in cMBL: any need to revise the current guidelines? Leukemia 26: 1703–1707.
- 8. Dagklis A, Fazi C, Sala C, Cantarelli V, Scielzo C, et al. (2009) The immunoglobulin gene repertoire of low-count chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis is different from CLL: diagnostic implications for clinical monitoring. Blood 114: 26–32.
- 9. Swerdlow SH (2008) WHO classification of tumours of haematopoietic and lymphoid tissues, fourth edition. Lyon, France: International Agency for Research on Cancer. 439p.
- 10. Nieto WG, Teodosio C, López A, Rodríguez-Caballero A, Romero A, et al. (2010) Primary Health Care Group of Salamanca for the Study of MBL: Non-CLL-like monoclonal B-cell lymphocytosis in the general population: prevalence and phenotypic/genetic characteristics. Cytometry B ClinCytom 78 Suppl 1S24–34.
- 11. Sánchez ML, Almeida J, Vidriales B, López-Berges MC, García-Marcos MA, et al. (2002) Incidence of phenotypic aberrations in a series of 467 patients with B chronic lymphoproliferative disorders: basis for the design of specific four-color stainings to be used for minimal residual disease investigation. Leukemia 16: 1460–1469.
- 12. Nieto WG, Almeida J, Romero A, Teodosio C, López A, et al. (2009) Increased frequency (12%) of circulating chronic lymphocytic leukemia-like B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach. Blood 114: 33–37.
- 13. Kalina T, Flores-Montero J, van der Velden VH, Martin-Ayuso M, Böttcher S (2012) EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols. Leukemia 26: 1986–2010.
- 14. Quijano S, López A, Rasillo A, Sayagués JM, Barrena S, et al. (2008) Impact of trisomy 12, del(13q), del(17p), and del(11q) on the immunophenotype, DNA ploidy status, and proliferative rate of leukemic B-cells in chronic lymphocytic leukemia. Cytometry B ClinCytom 74: 139–149.
- 15. González D, Balanzategui A, García-Sanz R, Gutiérrez N, Seabra C, et al. (2003) Incomplete DJH rearrangements of the IgH gene are frequent in multiple myeloma patients: immunobiological characteristics and clinical implications. Leukemia 17: 1398–1403.
- 16. van Dongen JJ, Langerak AW, Brüggemann M, Evans PA, Hummel M, et al. (2003) Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98–3936. Leukemia 17: 2257–2317.
- 17. Rossi D, Rasi S, Fabbri G, Spina V, Fangazio M, et al. (2012) Mutations of NOTCH1 are an independent predictor of survival in chronic lymphocytic leukemia. Blood 119: 521–529.
- 18. Costa ES, Pedreira CE, Barrena S, Lecrevisse Q, Flores J, et al. (2010) Automated pattern-guided principal component analysis vs expert-based immunophenotypic classification of B-cell chronic lymphoproliferative disorders: a step forward in the standardization of clinical cytometry. Leukemia 24: 1927–1933.
- 19. Rawstron AC (2009) Monoclonal B-cell lymphocytosis. Hematology Am Soc Hematol Educ Program: 430–439.
- 20. Kostareli E, Smilevska T, Stamatopoulos K, Kouvatsi A, Anagnostopoulos A (2008) Chronic lymphocytic leukaemia: an immunobiology approach. Srp Arh Celok Lek 136: 319–323.
- 21. Calin GA, Ferracin M, Cimmino A, Di Leva G, Shimizu M, et al. (2005) A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 353: 1793–1801.
- 22. Schwaenen C, Nessling M, Wessendorf S, Salvi T, Wrobel G, et al. (2004) Automated array-based genomic profiling in chronic lymphocytic leukemia: development of a clinical tool and discovery of recurrent genomic alterations. Proc Natl Acad Sci USA 101: 1039–1044.
- 23. Fabbri G, Rasi S, Rossi D, Trifonov V, Khiabanian H (2011) Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation. J Exp Med 208: 1389–1401.
- 24. Zenz T, Mertens D, Dohner H, Stilgenbauer S (2011) Importance of genetics in chronic lymphocytic leukemia. Blood Rev 25: 131–137.
- 25. Kern W, Bacher U, Haferlach C, Dicker F, Alpermann T, et al.. (2012) Monoclonal B-cell lymphocytosis is closely related to chronic lymphocytic leukaemia and may be better classified as early-stage CLL. Br J Haematol doi: 10.1111/j.1365–2141.2011.09010.x. In press.
- 26. Lanasa MC, Allgood SD, Slager SL, Dave SS, Love C, et al. (2011) Immunophenotypic and gene expression analysis of monoclonal B-cell lymphocytosis shows biologic characteristics associated with good prognosis CLL. Leukemia 25: 1459–1466.
- 27. Vardi A, Dagklis A, Scarfò L, Jelinek D, Newton D, et al.. (2013) Immunogenetics shows that not all MBL are equal: the larger the clone the more similar to CLL. Blood 2013 Apr17 [Epub ahead of print]
- 28. Del Giudice I, Rossi D, Chiaretti S, Marinelli M, Tavolaro S, et al. (2012) NOTCH1 mutations in +12 chronic lymphocytic leukemia (CLL) confer an unfavorable prognosis, induce a distinctive transcriptional profiling and refine the intermediate prognosis of +12 CLL. Haematologica 97: 437–441.
- 29. Rawstron AC, Bennett F, Hillmen P (2007) The biological and clinical relationship between CD5+23+ monoclonal B-cell lymphocytosis and chronic lymphocytic leukaemia. Br J Haematol 139: 724–729.
- 30. Shanafelt TD, Kay NE, Call TG, Zent CS, Jelinek DF, et al. (2008) MBL or CLL: which classification best categorizes the clinical course of patients with an absolute lymphocyte count >or = 5×10(9) L(-1) but a B-cell lymphocyte count <5×10(9) L(-1)? Leuk Res 32: 1458–1461.
- 31. Shanafelt TD, Ghia P, Lanasa MC, Landgren O, Rawstron AC (2010) Monoclonal B-cell lymphocytosis (MBL): biology, natural history and clinical management. Leukemia 24: 512–520.
- 32. Agathangelidis A, Darzentas N, Hadzidimitriou A, Brochet X, Murray F, et al. (2012) Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies. Blood 119 (19): 4467–4475.
- 33. Chu CC, Catera R, Hatzi K, Yan XJ, Zhang L, et al. (2008) Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA. Blood 112: 5122–5129.
- 34. Martin T, Duffy SF, Carson DA, Kipps TJ (1992) Evidence for somatic selection of natural autoantibodies. J Exp Med 175: 983–991.
- 35. Tobin G, Thunberg U, Karlsson K, Murray F, Laurell A, et al. (2004) Subsets with restricted immunoglobulin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia. Blood 104: 2879–2885.
- 36. Arcaini L, Zibellini S, Passamonti F, Rattotti S, Lucioni M, et al. (2009) Splenic marginal zone lymphoma: Clinical clustering of immunoglobulin heavy chain repertoires. Blood Cells Mol Dis 42: 286–291.
- 37. Buhler A, Zenz T, Stilgenbauer S (2010) Immunoglobulin heavy chain variable gene usage and (super)-antigen drive in chronic lymphocytic leukemia. Clin Cancer Res 16: 373–375.
- 38. Lenze D, Berg E, Volkmer-Engert R, Weiser AA, Greiner A (2006) Influence of antigen on the development of MALT lymphoma. Blood 107: 1141–1148.
- 39. Casabonne D, Almeida J, Nieto W, Romero A, Fernández-Navarro P, et al. (2012) Common infectious agents and monoclonal B-cell lymphocytosis: a cross-sectional epidemiological study among healthy adults. Plos One 7(12): e52808 doi:10.1371/journal.pone.0052808.
- 40. Almeida J, Nieto WG, Teodosio C, Pedreira CE, López A, et al. (2011) CLL-like B-lymphocytes are systematically present at very low numbers in peripheral blood of healthy adults. Leukemia 25(4): 718–22.