Chronic lymphocytic leukemia (CLL) has traditionally been viewed as a neoplastic proliferation of naive B cells of the mantle zone, but this view is now changing, with reports of somatically mutated immunoglobulin (Ig) variable heavy chain (VH) genes in CLL. 1, 2 Recently, Hamblin et al1 and Damle et al2 have independently demonstrated that CLL comprises 2 subsets with either mutated or unmutated VH genes. The mutated CLL cases had a more favorable prognosis and required less treatment than the unmutated cases. Both studies postulated that CLL cells might originate from 2 different stages of B-cell development, ie, pre-or post-GC (germinal center) B cells. Moreover, Damle et al found a strong correlation between VH gene mutational status and CD38 expression in CLL, where unmutated cases displayed a higher percentage of CD38 cells (30%) than mutated cases (30%). 1 Damle et al proposed that both VH gene mutational status and CD38 expression could be used as novel prognostic indicators of clinical outcome in CLL. But in a follow-up study by Hamblin et al, 3 they could not confirm any association between Ig mutational status and CD38 expression, although a significantly poorer prognosis was observed among the cases with more than 30% CD38. 3 We have analyzed the VH gene mutational status in 107 B-cell CLL (B-CLL) cases, and moreover, we have evaluated the CD38 expression in 48 of these cases to further clarify the correlation between VH gene mutations and CD38 expression. DNA was extracted mainly from lymph nodes and peripheral blood lymphocytes, and VH gene family–specific polymerase chain reaction (PCR) amplification was performed as previously described. 4 The majority of samples were sequenced directly using an automated DNA sequencer (ABI 377, Applied Biosystems, Foster City, CA), and the nucleotide sequences were compared to the BLASTN and V-BASE databases. Less than 98% homology to the corresponding germline gene was defined as a mutated VH gene. In a subset of cases, the surface expression of CD38 was evaluated using 2-color flow cytometry and direct conjugate antibodies (anti-CD19–FITC/anti-CD38–PE). We amplified and sequenced 115 clonal Ig rearrangements in 107 B-CLL cases, where 8 cases displayed 2 different Ig rearrangements within the same tumor sample. In accordance with previous studies, overexpression of the VH1 family (31.3%) and, particularly, the VH1-69 gene (16.5% of all rearrangements detected) was found. 1, 2, 4 Forty-eight cases (44.9%) showed mutated VH genes, whereas 59 cases (55.1%) were unmutated. The VH1-69 gene was exclusively expressed in unmutated cases with a high frequency (28% of unmutated VH genes), and the VH3-21 gene was mainly found expressed in the mutated cases (19% of mutated, as opposed to 3% of unmutated, VH genes). Interestingly, 1 of 8 cases displaying 2 VH rearrangements showed discrepancy in the mutation pattern between the 2 VH genes (94.4% versus 100% homology). Analysis of distribution of replacement (R) and silent mutations within the complementarity determining regions (CDRs) and framework regions (FRs) using the algorithm of Chang and Casali5 showed that 4 cases had significant clustering of R mutations in the CDRs and scarcity of R mutations in the FRs. In addition, 3 cases displayed clustering of R mutations within the CDRs, and in 6 cases evidence for preservation of the FRs was found. Thus in contrast to previous reports, only a minority of our CLL cases showed evidence of antigen-driven selection.

The CD38 expression was analyzed in a subset of 48 CLL patients, where …