A Single Cell Analysis of Immunoglobulin Genes in Chronic Lymphocytic Leukemia (CLL): Evidence of Intraclonal Diversification Associated with Progressive Somatic Mutations and Progressive Antigen Drive in Immunoglobulin Heavy and Light Chains.

Somatic mutations of immunoglobulin genes characterize mature memory B cell populations, and intraclonal B cell diversification is typically associated with expansion of B cell clones with greater affinity for antigen (antigen drive). Approximately half of patients with chronic lymphocytic leukemia (CLL) have malignant CLL cell populations that express immunoglobulin genes with somatic mutations. Recently, analysis of immunoglobulin gene utilization in CLL patients with unmutated immunoglobulin genes revealed multiple distinct sets of stereotyped B cell receptors in some CLL patients; this suggested that antigen may promote CLL development and/or progression. Evidence for a role of antigen in intraclonal CLL cell diversification in patients with mutated immunoglobulin genes has not been previously presented. Earlier, we performed a single cell analysis of immunoglobulin heavy and light chains from CLL patients and identified a CLL patient with mutated immunoglobulin genes and evidence of multiple related clones of CLL cells. We reported empiric construction of a tree of related clones from this CLL patient using data from the single cell analysis of immunoglobulin heavy and light chains (Blood102:666a, 2003). In the current analysis, we determined whether the clonally related populations of CLL cells from this patient exhibited evidence of antigen drive. We used the method of Lossos et al. (J Immunol165:5122, 2000) to assess the effect of somatic mutations on the ratio of replacement and silent amino acid changes in the framework and antigen binding regions (CDRs) of the immunoglobulin heavy and light chains from each oligoclonal population. There was a progressive increase and decrease, respectively, in the ratios of replacement to silent amino acid changes in the CDR and framework regions of immunoglobulin heavy chains from CLL cell clones that were descendants in the tree established by our earlier analysis. The amino acid changes were consistent with an antigen driven progression of clonally related CLL cell populations. The progressive amino acid changes in the CDRs and framework regions of the immunoglobulin heavy chains were associated with progressive improvements in the p values associated with these changes [range of p = 0.2273 for CDR analysis of initial clone in tree (clone A) to p = 0.0253 for one of final descendant clones in tree (clone N)]. Analysis of immunoglobulin light chains from these oligoclonal populations of CLL cells was consistent with an antigen driven process in all clones but did not show progressive improvements in clonal descendants (p = 0.0028 and p = 0.0043 for clones A and N, respectively, for CDR analysis). Taken together, these studies suggest that CLL cells can undergo intraclonal diversification after malignant transformation. These studies also support the hypothesis that development and progression of CLL are dependent on interactions of antigen with immunoglobulin receptors.