Intraclonal homogeneity of clonotypic immunoglobulin M and diversity of nonclinical post-switch isotypes in multiple myeloma: insights into the evolution of the myeloma clone.

PURPOSE: The transformation status and role of clonotypic pre-switch IgM in the evolution of malignant post-switch multiple myeloma (MM) cells is unclear. In this study, we determined the differentiation stage within the B lineage of clonotypic cells from malignant and nonclinical isotype pools by analyzing the frequency and intraclonal diversity of members within each isotype pool.
RESULTS: Immunoglobulin VDJ transcripts were amplified from peripheral blood cells of seven patients with a hemi-nested reverse transcription-PCR with complementarity determining region 1 (CDR1)-specific and constant region primers. Of the 1951 clones screened by patient CDR2/3-specific PCR, 356 of these were sequenced. Intraclonal homogeneity was observed in pre-switch transcripts from four of four informative patients. Transcripts from the IgM pool were relatively frequent in two of four informative patients. Cellular limiting dilution analysis indicated 0.4-25% of peripheral blood mononuclear cells expressed clonotypic IgM for 6 of 15 samples tested. By contrast, significant intraclonal diversity was observed in the nonclinical IgA pool of 1 patient. A genealogical tree of IgA sequences was constructed showing ongoing clonal diversification from sequences with close homology to the germ-line V gene to those resembling the PC sequence. Furthermore, some clones exhibited complete homology with tumor VDJ sequence, plus extra mutations, suggestive of a parallel clonal arm that remains responsive to an antigenic stimulus.
CONCLUSIONS: Detection of intraclonal diversity in the post-switch nonclinical isotype pool suggests that remnants of the parent B-cell clone coexist with malignant clonal precursors. The presence of intraclonal homogeneity in the pre-switch IgM pool supports the idea that pre-switch MM cells play a role in malignant events within the MM clone.