Monoclonal B Cell Lymphocytosis Exhibits Biologic Diversity Similar to Chronic Lymphocytic Leukemia.

Background: Monoclonal B cell lymphocytosis (MBL) is a pre-clinical syndrome characterized by small accumulations of monoclonal B lymphocytes in the peripheral blood. MBL have an immunophenotype similar to CLL: CD5⁺, CD19⁺, CD20^lo, CD23⁺, CD79b^lo, sIg^lo. The biologic characteristics and clinical implications of MBL remain unclear. We hypothesized that MBL have a biology similar to CLL and represent a pre-neoplastic state of CLL. Characterization and longitudinal evaluation of MBL may provide insight into mechanisms of CLL leukemogenesis.

Methods: We defined MBL as a population of CD5⁺, CD20^lo cells that were kappa/lambda restricted and comprised at least 2% of the CD19⁺ peripheral B cell compartment. Persons with MBL were ascertained by flow cytometric screening of peripheral blood from unaffected members of CLL kindreds. Flow cytometric analysis and fluorescence-activated cell sorting (FACS) were used to determine the surface immunophenotype and molecular characteristics of MBL. MBL cells were bulk-sorted and analyzed using FISH for loci associated with clinical CLL [13q14.3 (D13S319), 17p13.1 (TP53), 11q22.3 (ATM), and 12p11.1-q11 (enumeration probe)]. Single MBL cells were sorted and genomic DNA analyzed by PCR to determine immunoglobulin heavy and light chain sequences.

Results: Twelve out of 113 (11%) unaffected family members were found to have MBL. Seven of these individuals provided additional blood for MBL characterization. All 7 MBL subjects had normal complete blood counts and leukocyte differentials. Six of the 7 MBL subjects had MBL counts less than 150 cells/μL. All MBL samples had immunophenotypes similar to CLL. One MBL sample was CD38⁺/Zap70⁺, one was CD38⁺/Zap70⁻, one was CD38⁻/Zap70⁺, and 4 samples were CD38⁻/Zap70⁻. The ratio of median fluorescence intensity of CD69:CD71 was low (<1.0) in 6 of the 7 MBL samples, but was 2.8 in the one CD38⁺/Zap70⁺ case, consistent with an activated membrane surface phenotype. Next, MBL cells were sorted as single cells and genomic DNA was amplified for direct sequencing of immunoglobulin heavy and light chains. This analysis has been completed in 4 of 7 individuals. Immunoglobulin heavy chain variable (IgV_H) region gene usage mirrored that of typical CLL and included examples of IgV_H mutated and unmutated sequences. Some MBL were monoclonal, some consisted of related antigen-driven oligoclonal populations, and others consisted of unrelated oligoclonal populations. For example, one person showed three unique clones: a mutated VH4-59 clone (38% of all MBL cells), a mutated VH4-34 clone (5%), and an unrelated unmutated VH4-34 clone (57%). FISH analyses showed mono- or biallelic deletion of 13q14 in four of seven samples. No other loci were deleted or amplified in these 7 MBL subjects. Four individuals provided a second sample of blood for longitudinal MBL analysis. The size of the MBL clone expanded in 3 of these subjects and was unchanged in the 4^th subject.

Conclusions: This cohort of familial MBL recapitulated much of the biologic diversity of clinical CLL. By several different phenotypic measures, the MBL phenotype was similar to typical CLL. To our knowledge, this is the first report of CD38⁺, Zap70⁺, and IgV_H unmutated MBL. In contrast to most CLL, we found that many MBL consisted of more than one clonal population. Likewise, we identified evidence of ongoing antigen-driven intraclonal diversification in MBL.