Evidence for progenitors of chronic lymphocytic leukemia B cells that undergo intraclonal differentiation and diversification

Peripheral blood mononuclear cells from five patients with IgG⁺ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone- specific Ig H chain variable region gene mRNA transcripts linked to Cμ and/or Cα. This was assessed by (1) comparing the lengths of portions of the V(H)DJ(H) of the IgG⁺ CLL clones with those of the μ and α isotype- expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the μ and α isotype- expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with Cγ-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG⁺ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using Cμ- and/or Cα-specific primers, strongly suggesting clonal relatedness. These CLL-related μ- and α-expressing cDNA were present in greater amounts than unrelated (non-CLL) μ- and α-expressing cDNA from normal B cells that used genes of the same V(H) family. When the sequences of these CLL-related Cμ- and Cα-expressing cDNA were compared with those of the IgG⁺ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the μ- and α-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM⁺ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG⁺ B-CLL and that these cells may differentiate, accumulate V gene mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.