Single-cell analysis reveals oligoclonality among 'low-count' monoclonal B-cell lymphocytosis

Abstract

Monoclonal B-cell lymphocytosis (MBL) is a preclinical hematologic syndrome characterized by small accumulations of CD5(+) B lymphocytes. Most MBL share phenotypic characteristics with chronic lymphocytic leukemia (CLL). Although some MBL progress to CLL, most MBL have apparently limited potential for progression to CLL, particularly those MBL with normal absolute B-cell counts ('low-count' MBL). Most CLL are monoclonal and it is not known whether MBL are monoclonal or oligoclonal; this is important because it is unclear whether MBL represent indolent CLL or represent a distinct premalignant precursor before the development of CLL. We used flow cytometry analysis and sorting to determine immunophenotypic characteristics, clonality and molecular features of MBL from familial CLL kindreds. Single-cell analysis indicated four of six low-count MBL consisted of two or more unrelated clones; the other two MBL were monoclonal. 87% of low-count MBL clones had mutated immunoglobulin genes, and no immunoglobulin heavy-chain rearrangements of V(H) family 1 were observed. Some MBL were diversified, clonally related populations with evidence of antigen drive. We conclude that although low-count MBL share many phenotypic characteristics with CLL, many MBL are oligoclonal. This supports a model for step-wise development of MBL into CLL.

Figure 1. MBL Identification by flow cytometry

In A, the MBL flow cytometric gating strategy from PBMC is shown. MBL gating steps included a forward by side scatter plot, with low forward and side-scatter events gated, a CD19 by CD3 / CD14 / CD16 (dump antibodies to exclude T cells, monocytes, NK cells and granulocytes) plot, with CD19⁺ and dump negative events gated, and a CD20 by CD5 plot, with CD5⁺CD20^lo MBL gated. In this sample, immunoglobulin light chain κ restriction was observed. Further confirmation of MBL was performed and indicated that MBL were (B) CD23⁺, CD27⁺, and CD79b^lo.

Figure 2. MBL subject with intraclonal diversification

In A, genealogical analysis of related MBL subgroups based on sequences from 37 single MBL cells from subject 0602, and analysis of the rearranged immunoglobulin V_H3-07 heavy and V_L1b light chains from each MBL cell subgroup. Letters A — L represent individual subgroups of MBL cells with a related immunoglobulin heavy chain gene and light chain rearrangement. The number of individual cells identified from each subgroup is shown in orange to the right of the subclone sequence. GL represents the germline V_H3-07 sequence. Empty circles with dashed lines (representing multiple potential clones) and circles with dashed lines labeled H1, H2 and H3 (individual clones) represent hypothetical transitional MBL clones that were not observed in the analysis. In B, antigen drive analysis with p values calculated using the method of Lossos et al (2000) (23). In brief, the ratio of amino acid replacement (R) to synonomous (S) nucleotide base changes is compared in both the framework (Fr) and complementarity determining region (CDR) to determine if the observed mutations are random or antigen driven (21).