Molecular analysis of rearranged VH genes during B cell chronic lymphocytic leukemia: intraclonal stability is frequent but not constant.

Several genetic mechanisms have been shown to diversify the expressed antibody repertoire of committed B lymphocytes. These include V gene replacement, ongoing gene rearrangement and somatic hypermutation. These mechanisms may be operational at discrete points in the B cell differentiation pathway and generate idiotype diversity in various malignant B cell tumors. In particular, V region mutations have been established as a major mechanism of tumor escape from anti-idiotype immunotherapy in some lymphoma. On the other hand, previous studies on a few selected cases have shown that this mutation process does not affect the B cell clone during chronic lymphocytic leukemia. However, to what extent this intraclonal stability is a general phenomenon during B cell CLL is not clear. Therefore, we randomly selected 6 patients suffering from classical B cell CLL (sIgM (+), CD5 (+), CD19 (+)) at different stages of the disease and analysed the intraclonal variability of the expressed variable region of the heavy chain (VH). After PCR amplification of the cDNA corresponding to the rearranged VDJ regions, the products were cloned and sequenced. In five cases, multiple clone analysis did not show any intraclonal variability whatever the stage of the disease. Furthermore, in a single case, this intraclonal stability was confirmed during a three year period of time when the disease progressed. The sixth case behaved differently since we found multiple nucleotide substitutions, apparently accumulating as the malignant clone expanded. Besides the theoretical difficulties that these changes can induce during immunotherapy, two findings merit further discussion: 1) the distribution of the ongoing mutations affecting the VH region was not suggestive of an antigen driven selection, 2) this intraclonal variability was specific for the VH region, since the VL region showed no intraclonal variation.