The monoclonal Ig expressed on CLL cells was considered to be identical in all cells of the leukemic clone in a CLL patient. However, intraclonal VDJ gene diversification was recently reported in CLL B cells using SSCP analysis and sequencing of Ig PCR products exhibiting altered mobility. We used a different approach, by cloning directly monoclonal VHDHJH PCR bands from CLL patients and sequencing a large number of clones. Peripheral blood from five patients with typical CLL was used to extract DNA.. In one patient DNA from bone marrow and a lymph node biopsy was also analysed. PCR was performed with VH family specific primers and a consensus JH primer for 30 cycles. Monoclonal bands were ligated to TOPO vector and 25 to 70 insert containing clones were sequenced from both orientations. Sequenses contigs were aligned with DNASTAR and were compared with the germ line counterpart using BLAST and IMGT data bases. Intraclonal base variation was observed in 8–33% of the clones in all five patients. One to five base point-mutations /clone were found and there were mostly transitions (in 79% of mutations). In one patient with two monoclonal Ig sequences, (one hypermutated V3-7 and one unmutated VH3-23) intraclonal variation was observed in 69% and 8% of the clones respectivelly. In two patients clones with partially shared mutations were observed indicating the further diversification of a parental subclone. In the patient that VHDHJH clones from blood/bone marrow/lymph node were obtained, a difference in the frequency of diversified clones was noted. All samples were obtained from the patient within one month to exclude the possibility of time related mutations in the monoclonal Ig of the patient. In the patient’s VH1-69 unmutated sequence, 19 of 64 (29,6%) of bone marrow derived clones had additional mutations but they were found in only 12 of 71 (16%) peripheral blood derived clones. Five of 53 (9,4%) lymph node derived clones had additional point mutations. Low R/S ratio and targeting of FR rather than CDR regions is against the hypothesis of antigen driven somatic mutations in a fraction of the CLL cells in a given patient. The difference in diversification rate between different tissues in the same patient raises the question of the role of the patient microenvironment/stroma in partcipating in these mutations observed in CLL subclones. With the analysis of large number of clones it is possible that all CLL patients may have at least some degree of intaraclonal VHDHJH gene diversification.

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