Clonotypic IgG B Cells in Waldenstrom Macroglobulinemia Cells Are Derived from Multiple Post-Transformed B Cells That Have Undergone Isotype Switch Recombination.

Clonotypic B cells of Waldenstrom Macroglobulinemia (WM) are characterized as CD20⁺138⁻ sIgM⁺sIgD⁺ cells that are not restricted to CD27 memory marker expression. Clonotypic VDJ sequences show VH3/JH4 gene bias, exhibit intraclonal homogeneity and do not undergo class switch recombination (CSR). To better understand the failure of CSR in WM, switch (S) region analysis, activation induced cytidine deaminase (AID) expression and CD40L/IL-4 activated clonal CSR were studied in comparison to IgM monogammopathy of undetermined significance (MGUS). Large internal deletions of S_μ that could hinder CSR were determined by analysis of CDR2/S_μ fragments amplified from genomic DNA of clonotypic WM or MGUS B cells. Deletions were found in only 1/12 WM and 0/4 IgM MGUS, suggesting that large deletions of S_μ are not common in WM or IgM MGUS. Sequence analysis of S_μ upstream regions revealed striking differences between WM and IgM MGUS. By comparing the 1.6 kb sequences upstream of S_μ tandem repeats, we found that WM clones exhibit less mutation (0–2 bp/1.6 kb or 0.57x10^{− 3} bp, n=11) than IgM MGUS (0–7 bp/1.6 kb or 2.5x10^{− 3} bp, n=4, p=0.001). The MGUS clones containing frequently mutated switch region also exhibited intraclonal diversity of IgM VDJ. These differences exclude most IgM MGUS from a precursor relationship with WM and may help to identify those less frequent IgM MGUS that are at risk of progression to WM. Only 2/17 WM and 0/5 IgM MGUS constitutively expressed AID but it can be induced by CD40L/IL-4 activation as shown in 4/4 WM and 3/3 IgM MGUS. Single cell analysis showed that both clonotypic and non-clonotypic B cells accounted for AID induction. Splice variants are colocalized with full-length transcripts in some individual cells from WM, IgM MGUS and normal donors. Thus, AID splice variants are generally present in activated B cells. Using enhanced PCR amplification protocols, clonotypic CDR2/C_γ transcripts were detectable post-stimulation in 2/4 WM and 2/3 IgM MGUS, suggesting that WM and IgM MGUS B cells are capable of CSR. For 2 WM activated in vitro, we detected clonal IgG transcripts, having variable-sized CDR2/S_γ 1 fragments and diversity of clonotypic S_μ/S_γ junctions. This indicates that clonotypic CSR occurred multiple times within the WM tumor population. Although this implies that persistent CSR occurs and although clonotypic IgM B cells were frequent (16%) in cultures, clonotypic IgG producing WM B cells were very rare, with a frequency of only 0.01%. Clonotypic IgG exhibited intraclonal homogeneity of the clonotypic VDJ, with a sequence identical to that of the clonotypic IgM VDJ, suggesting that clonotypic CSR is a post-transformation event. Clonotypic IgG may arise through CSR occurring in vitro, through clonal expansion of pre-existing post-switch WM B cells or both. Overall, our studies show that initial B cell activation as determined by AID induction is normal and that intact CSR machinery is functional in WM, although clonotypic CSR occurs at very low frequency. For the majority of WM B cells, CSR does not occur even when stimulated in vitro, suggesting that the WM cell is constitutively unable to undergo CSR or being prevented from CSR. Further, since mutations in upstream S_μ are found in most IgM MGUS, making them ineligible as WM progenitors, switch sequence analysis may help to identify those IgM MGUS at risk of progression.