Complex Paterns of Monoclonal Ig Diversification in B-CLL Cells from Different Tissue Compartments.

It has recently been demonstrated that a considerable subset of CLL patients (>50%) displays intraclonal diversification in their monoclonal Ig sequence, suggesting that the mechanism of ongoing somatic mutations is retained in a significant proportion of B CLL cells. The complexity of studying this phenomenon relies on the enclaved risk of misinterpreting superimposed mutations originating from experimental artifacts. We are still lacking a more detailed picture of inraclonal diversification regarding its frequency and developmental pattern in disease compartments other than blood. In order to clarify some of these questions, we analyzed a total of 675 Ig clones from 11 CLL patients. DNA was extracted from blood in 6 patients (1st group: pt.1 to 6) or from blood/marrow (BM)/lymph nodes (LN) obtained at the same in 5 patients (2^nd group: pt.7-11). PCR was performed for 40 cycles with Taq Platinum polymerase and leader or FR1 VH1-6 primers and a consensus JH primer. Monoclonal PCR products were ligated to TA vector and 20 to 70 clones were sequenced. from both orientations. Polymerase mediated base error was 4.4x10⁻⁴ after 40 PCR cycles. Base differences observed in two or more clones out of 20, were verified twice and assigned as true intraclonal diversification. This was observed in 6 (pt 2, 6, 7, 9, 10, 11) out of 11 pts (54.5%). Pts 2, 6, 10, 11 were mutated and pt. 7, 9 unmutated in their Ig sequence. Intraclonal diversification was observed in pt. 2 and 6, whereas pt.7, 9, 10, 11 in the 2nd group, displayed intraclonal diversification in some or in all compartments. Base point mutations that were unique in one clone only were observed at the polymerase mediated error rate and were not verified by a second idependent experiment. In the 2nd group pt. 8 displayed lack of diversification both in blood and marrow. Pt.7 displayed diversification only in BM clones (2/64, 3.1%) but not in 71 blood derived or in 53 LN derived clones. Pt. 9 had diversified clones in blood only (2/26, 7.6%) and no diversified clones in 25 BM clones. Pt.10 had diversified clones both in blood and marrow with a distinct pattern: in blood (62 clones) 4 different patterns of diversification were observed in 54 clones (84%); 17/54 were evolving clones. In BM two of the 4 patterns of diversification present in blood were observed in 28/57 clones (51.7%); ten were evolving clones. In pt.11, no diversified clones were present in blood (50 clones) whereas marrow had 4/60 diversified clones (6,66%, two patterns of diversification) and LN had 20/40 diversified clones (50%, two patterns of diversification); 8/20 diversified were evolving clones. The patterns of diversification were different bettween LN and BM in this patient. Base changes in 110 diversified clones were in FRs rather than CDRs (16:1) and resulted in S(silent) rather than R(replacements), S/R 2:1. In conclusion, intraclonal diversification must be considered true if identical base substitutions are observed in minimum two clones. Pattern and frequency are not identical in B CLL cells from different sites (blood vs BM vs LN). Preferance of FRs v CDRs and SvR substitutions implies that diversification procces is not antigen driven. The presence of a functional mechanism of ongoing somatic mutations in leukemic clones and the ability of diversification to target with altered frequency/patterns various disease compartments implies a role of tissue millieu in CLL clone evolution.