Intraclonal homogeneity of clonotypic immunoglobulin M and diversity of nonclinical post-switch isotypes in multiple myeloma: insights into the evolution of the myeloma clone.
OBJECTIVE
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.
Taylor
Brian J
BJ
Department of Oncology, University of Alberta, Edmonton, Alberta, T6G 1Z2 Canada.
Pittman
Julie A
JA
Seeberger
Karen
K
Mant
Michael J
MJ
Reiman
Tony
T
Belch
Andrew R
AR
Pilarski
Linda M
LM
D001483:Base Sequence; D002454:Cell Differentiation; D003001:Cloning, Molecular; D015321:Gene Rearrangement; D006801:Humans; D017578:Immunoglobulin Class Switching; D007075:Immunoglobulin M / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000235:genetics* / Q000276:immunology*; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D007075:Immunoglobulin M