Study of IGH sequences in low-count monoclonal B-cell lymphocytosis

Abstract

Low-count monoclonal B-cell lymphocytosis (MBLlo) is defined by the presence of <0.5 × 10! clonal B-cells/L in blood in the absence of any other symptoms or signs of a neoplastic lymphoproliferative disorder. It is still unclear whether MBLlo represents a pre- malignant state prior to chronic lymphocytic leukemia (CLL) or it relates to a physiological immunosenescent process. The molecular and biochemical characteristics of the B-cell receptor (BCR) including the specific immunoglobulin heavy-chain (IGH) sequences have been related to disease behavior in CLL. In MBLlo, few studies have been reported of IGH sequences on the clonal B-cells, mostly due to their low counts in blood. Here, we set up a workflow adapted to the low MBLlo clonal B-cell numbers aimed at more efficiently characterize genetically and molecularly the IGHV rearrangements of the BCR of MBLlo cases. We also compare genetic and molecular characteristics of the BCR of MBLlo vs MBLhi and CLL B-cell clones, and their impact on the behavior of MBLlo clonal B-cells during follow-up. MBLlo clonal B-cells were isolated from blood of otherwise healthy individuals by high- sensitive flow cytometry. Two different multiplex PCR protocols for IGHV sequencing were applied, depending on the number of MBLlo clonal B-cells available, using a threshold of 50,000 cells for protocol selection. The new workflow set up allowed us to increase the number of MBLlo cases included in out cohort (N=34), for subsequent comparison of IGHV sequences to two MBLhi (N=73) and CLL (N=138) cohorts. The IGHV3-7 gene sequence was more frequently found in MBLlo subjects, whereas IGHV3-23 was highly represented in MBLhi patients. No significant differences were reported in D segments, while IGHJ4 was significantly more frequent in MBLlo and IGHJ6 in CLL. Compared to other J-gene segments, clonal B-cells with IGHJ6 show a more immature origin and present longer HCDR3 sequences. Nevertheless, this BCR characteristic showed no significant impact in the behavior of the MBLlo clone during follow-up; however a tendency towards a greater size of MBLlo clones was observed among IGHJ6 cases. The new workflow here proposed proved to be a useful tool to increase the efficiency of the characterization of the BCR sequences of MBLlo cases with low clonal B-cells numbers revealing unique BCR sequences vs MBLhi and CLL.