Molecular Characterization of Clonotypic VDJ Sequences in Waldenstrom’s Macroglobulinemia Suggests Independent Transformation Events in the Development of Biclonal B Cells.

Malignant B lineage cells in Waldenstrom’s macroglobulinemia (WM) express a unique clonotypic VDJ associated with IgM. Studies of WM patients revealed a frequent incidence of biclonal B cells (16%) as defined by the presence of two distinct clonotypic VDJ sequences. This is the first report to estimate the frequency of biclonality in WM. Four WM cases are reported: two with distinct IgM clones (WM1-19 and WM1-09), one with distinct IgM/IgA clones (WM1-18), and one with related but diversified IgM/IgG clones (WM10). In 2 cases (WM1-19 and WM10), the two clonotypic signatures were found to be abundant in bone marrow (BM) but less frequent in blood, reminiscent of monoclonal WM cases. In WM1-19, single cell analysis showed that only one partner VDJ was expressed per cell, excluding the possibility of aberrant biallelic rearrangements. The distribution ratio between the two tumor clones was 2:1, suggestive of their mutual role in the clinical manifestation. In the other 2 cases (WM1-09 and WM1-18), partner clones were shown by CDR3 fragment analysis to be anatomically distinct, with one BM clonotypic signature and one in blood. In these 2 patients, the BM clone was hypermutated (6.2% and 3.8%) while the blood clone was germline or minimally mutated (0% and 1.0%). Partner clones lacked intraclonal diversity and Ig class switching, characteristic of malignant WM clones, suggesting relatively frequent transformation events throughout B lineage differentiation. The biological events leading to the appearance of clones in two different anatomic sites and the clinical implications remain to be understood. CDR3 fragment analysis in the longitudinal studies of WM1-09 and WM1-18 suggested that the repertoire of blood B cells may recover some level of diversity after successive cycles of treatment (WM1-18), while the persistence of the monoclonal peak (WM1-09) likely reflects a tumor that does not respond to treatment. In WM10, biclonal IgM/IgG B cell clones shared common VDJ gene rearrangement. The IgG clone displayed a higher mutation rate than did the IgM clone (9.7% vs. 6.1%). Increased somatic hypermutation in IgG consists mostly of replacement mutations that are clustered within the CDR regions, strongly supporting a contention that the IgG clone has undergone affinity maturation. There are 11 point mutations in the IgM that are not present in the IgG, suggesting that the two clones are distinctly different and belong to different branches of the genealogical tree. These distinct mutational signatures suggest that biclonal IgM/IgG clones are unlikely derived from clonal evolution. Overall, our results suggest that for the four biclonal WM evaluated here, the partner B cell clones appear to have undergone separate transformation events in the development of biclonality. The extent to which each partner clone contributes to disease progression and death, whether separately or in synergy, is as yet unknown.