Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare disorder, which mostly affects middle-aged women and is associated with smoking.1 It is characterized by the persistent expansion of CD27+IgM+IgD+ B cells, the presence of circulating binucleated lymphocytes and increased IgM serum levels.1, 2, 3 Furthermore, PPBL is associated with HLA-DRB1*07 carriership and a supernumerary isochromosome +i(3q) in a small proportion of lymphocytes.3 Although the disorder is usually benign,4 ∼10% of patients present with mild splenomegaly and a few patients subsequently develop a mature B-cell malignancy, such as diffuse large B-cell lymphoma (DLBCL) or splenic marginal zone lymphoma (MZL).4 Despite an increasing number of described cases, the nature of the persistent lymphocytosis and its relation to mature B-cell malignancies remain poorly understood. Therefore, we subjected PPBL patients and healthy controls to immunoglobulin (Ig) repertoire and replication history analyses, and gene expression profiling (detailed Materials and Methods online).
Five patients (4 women, 1 man) were included with a moderate lymphocytosis of CD27+IgM+IgD+ B cells that was persistent for >1 year (Supplementary Table 1, Supplementary Figure 1a). These cells showed a normal Igκ/Igλ ratio, but abnormally low CD38 and CD21 expression levels (Supplementary Figure 1b). IGH-CDR3 immunospectratyping revealed polyclonal IGH rearrangements with a normal length distribution and in-frame selection (Supplementary Figure 1c). This was confirmed by detailed sequence analyses of 375 in-frame IGH gene rearrangements from sorted CD27+IgM+IgD+ cells. Similar to healthy controls, each clone yielded a unique sequence, and diverse IGHV, IGHD and IGHJ genes were used without signs for preferential pairing (Supplementary Figure 2). The IGHV1-2*04 allele that is associated with splenic MZL was used in only four rearrangements from three patients.5 Considering that 4/5 patients carried an IGHV1-2*04 allele in their germline DNA, it did not seem preferentially rearranged in PPBL cells. Finally, the IGH-CDR3 size distribution in IGH gene rearrangements of PPBL was similar to control natural effector and IgM-only memory B cells (Supplementary Figure 2f). Thus, PPBL cells showed a diverse Ig gene repertoire with normal IGH-CDR3 size selection and without obvious selection for IGHV gene use.
To study the nature of the lymphocytosis, we quantified the in vivo replication history with Ig kappa-deleting recombination excision circles.6 The observed proliferation of ∼16 cell divisions was significantly higher (P<0.05) than the replication histories of natural effector and IgM-only B cells of controls (Figure 1a). Despite the increased number of cell divisions, PPBL showed low levels of SHM, both in an Igκ CDR1 hotspot region and in IGHV genes (Figures 1b and c). These low SHM frequencies did not seem to result from impaired activity of activation induced cytidine deaminase or the error-prone Polymerase η, because targeting of RGYW/WRCY and WA/TW DNA sequence motifs in IGHV genes was normal (Supplementary Table 2).7 To study signs of antigenic selection, we determined the replacement/silent mutation ratios for framework (FR) and complementary determining regions (CDR), and compared these with the expected ratios using the BASELINe program.8 The FR of PPBL showed negative selection for replacement mutations similar to controls. In contrast, the CDR of PPBL did not show the positive selection of replacement mutations that was found in natural effector and IgM-only B cells from controls (Figure 1d). Thus, when compared with memory B cells from healthy controls, PPBL cells proliferated extensively, but showed unusually low SHM levels and limited signs of antigen-driven selection.
To obtain more insight into the origin and maturation status of PPBL cells, we performed genome-wide gene expression profiling in purified PPBL cells of four patients as well as five B-cell subsets from three healthy controls. Both normal and PPBL B-cell subsets showed high expression of genes encoding pan-B markers, including CD19, CD20, CD22, CD79A, CD79B and BAFF-R (Supplementary Table 3). The gene expression patterns for markers used to isolate the subsets (CD5, CD38, IgM and IgD) correlated well with protein expression. PPBL and control B-cell subsets were out of cell cycle as revealed by low MKI67 gene expression and absence of nuclear Ki67 protein (not shown). Furthermore, PPBL cells showed high expression of the genes encoding CD27, CD80, CD86, CD180 and TACI fitting with an activated memory B-cell profile.9 Supervised clustering of the subsets with the probe sets that showed the highest signal variation over all arrays (threshold 4; 643 probe sets) confirmed that PPBL B cells were more similar to control IgM+ memory B-cell subsets (distance, 0.25) than control naive B cells (distance, 0.55; Figure 1e). Still, PPBL B cells showed a distinct gene expression profile from control IgM+ memory B cells as revealed with unsupervised clustering (Figure 1f; Supplementary Table 4).
The expression levels of oncogenes that are typically involved in immature and mature B-cell malignancies showed large variation between individual donors of PPBL and control B-cell subsets, and no single oncogene was upregulated in all PPBL cases (Supplementary Table 3; Supplementary Figure 4). Two patients showed high BCL2 levels, but these were not caused by t(14,18), because these were only present in 1 out of 1.3 × 103 PPBL cells (Supplementary Figure 3).
We did not confirm previously observed deregulation of the TGFβ signaling pathway and the activator protein 1 complex.10, 11 Moreover, multiple genes encoding Fas signaling proteins were upregulated (Supplementary Figure 5). These findings do not support impaired apoptosis in PPBL. The discrepancy with previous studies is likely due to the fact that we analyzed purified B-cell subsets, providing a higher resolution to study differences in gene expression levels.
To identify novel immunophenotyping markers for PPBL cells, we selected eight differentially expressed genes between PPBL and control memory B cells that encoded membrane proteins and for which monoclonal antibodies were available (Supplementary Figure 6a). Of these, CD62L and CD73 expression levels were the most discriminative between PPBL and control memory B cells with flow cytometry (Supplementary Figure 6b). Although both markers showed a bimodal expression pattern on control memory B-cell subsets, their expression levels were uniformly low on all PPBL cells. Importantly, CD62L and CD73 were not downregulated in heavy smokers that were otherwise healthy (Figure 2a). Thus, the low levels of CD62L and CD73 seemed markers of PPBL rather than of heavy smoking.
CD73 and CD62L could not be used independently to fully discriminate PPBL cells from control B-cell subsets. Therefore, we applied principal component analysis to study whether the combined information of CD62L, CD73, CD38 and IgD expression levels would be sufficient for discrimination of PPBL from controls.12 In a 2D projection, PPBL was separated from controls mainly by CD73 and CD62L (principal component 1), whereas control natural effector and IgM-only B cells were separated mainly by IgD and CD38 (principal component 3; Figure 2b). Concluding, we showed that PPBL cells and control IgM+ memory B cells can be separated based on a limited set of membrane markers: CD38, IgD and the newly identified CD62L and CD73.
MyD88/TIRAP/IRAK4 signaling, probably downstream of Toll-like receptor 10 (TLR10), has been shown to be critical for homeostatic maintenance of CD27+IgM+IgD+ B cells.13 PPBL cells showed normal TLR1, and TLR6 expression, and significantly upregulated TLR10 expression (Figure 2c, Supplementary Table 3, Supplementary Figure 6a). Moreover, MYD88, IRAK1 and TLR-signaling target gene TNF were upregulated in PPBL compared with control IgM+ memory B cells (Supplementary Table 3, Supplementary Figure 4). These increased levels of TLR10 and downstream signaling components could make PPBL B cells more receptive to exogenous signals and thereby contribute to the polyclonal expansion.
To study how closely the PPBL phenotype resembles that of a mature B-cell malignancy, we performed immunophenotyping in four PPBL cases with the EuroFlow B-CLPD antibody panel.14 Multiparameter analysis was subsequently performed to compare the PPBL profile with those of nine diagnostic subgroups of mature B-cell malignancies. Principal component analysis revealed close clustering of all PPBL samples, implying that this is a truly unique biological entity. PPBL cells clustered separately from Burkitt lymphoma, follicular lymphoma, CD10+ DLBCL, hairy cell leukemia and chronic lymphocytic leukemia. Still, in the automatic population separator view, the 1 s.d. line of PPBL overlapped with lymphoplasmacytoid lymphoma (LPL) and CD10-DLBCL, and to a lesser extent with MZL and mantle cell lymphoma. The separation between PPBL and LPL was based mainly on the lower expression of CD200 and higher expression of CD305 (LAIR-1) in PPBL. Separation between PPBL and CD10-DLBCL was mainly based on the high expression levels of CD79b and IgM in PPBL. In contrast to healthy controls, CD62L contributed little to separation between PPBL and any of the analyzed malignancies (<10%). Thus, discrimination of PPBL from mature B-cell malignancies is based on different markers than healthy controls and is possible with currently implemented EuroFlow immunophenotyping protocols.14 This may be of value to discriminate between PPBL and rare biclonal malignancies with a seemingly normal Igκ/Igλ ratio.15 Interestingly, in a few described cases, the diagnosis of PPBL was complicated by DLBCL and splenic MZL, but not any other hematological malignancy.4 These observations suggest an origin from the same cell compartment and support the hypothesis that PPBL might precede B-cell malignancy development in rare cases.4
In this study, we showed for the first time that CD27+IgM+IgD+ cells in PPBL have undergone extensive proliferation, presumably in lymphoid tissues, such as the spleen. The high Ig diversity with absence of stereotypes and the distinctive immunophenotype of PPBL are suggestive of a different pathophysiology than in mature B-cell malignancies. Still, individual cases show some deregulated oncogene and tumor-suppressor expression, and might be prone to develop into a malignancy. The newly identified cell surface markers can prove valuable for diagnosis of PPBL and discrimination from B-cell malignancies.
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We thank I Pico and AA Tarique for technical support, S de Bruin-Versteeg for assistance with preparing the figures, Drs AW Langerak, VHJ van der Velden and M van der Burg for fruitful discussions and critical reading of the manuscript and the EuroFlow Consortium for kindly providing the EuroFlow tools and the data on mature B-cell malignancies. This work was supported by a grant from the Erasmus University Rotterdam (EUR-Fellowship to MCvZ).
The authors declare no conflict of interest.
MAB, JJMvD and MCvZ designed the experiments. CG-W, HJA and DdR provided conceptual advice. MAB and CG-W performed and analyzed most of the experiments. DdR and MCvZ contributed to data analyses. HJA, KPM-O, HJA, SB, AO provided material necessary for performing experiments and data analysis. MAB and MCvZ wrote the manuscript. CG-W, HJA, DdR, KPM-O, HJA, SB, AO and JJMvD commented on the manuscript.
Supplementary Information accompanies this paper on the Leukemia website
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Berkowska, M., Grosserichter-Wagener, C., Adriaansen, H. et al. Persistent polyclonal B-cell lymphocytosis: extensively proliferated CD27+IgM+IgD+ memory B cells with a distinctive immunophenotype. Leukemia 28, 1560–1564 (2014). https://doi.org/10.1038/leu.2014.77
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