Somatic mutations and intraclonal variations in the rearranged V(K) genes of B-non-Hodgkin's lymphoma cell lines

Three established Burkitt's lymphoma (BL) cell lines (Daudi, Raji and DG-75) and three B-non-Hodgkin's lymphoma (B-NHL) of other types (Pfeiffer, Farage and Toledo) were analyzed with respect to the presence of somatic point mutations in their rearranged immuno- globulin V(κ) genes. Two of the V(κ) sequences of BL and two of those of the B-NHL were heavily mutated (up to 11%), when compared with their closest germline variable region counterparts ('clonal mutations'). Only one of the six cell lines contained an unmutated germline V(κ) sequence. The clonal mutations have features characteristic of the mutation machinery operating in the course of the T- dependent immune response, such as a preference of mutations in purine bases, more transitions than transversions and targeting to CDR and to known 'hotspot' motifs. Sequence variations among different V(κ) PCR clones isolated from each of the cell lines ('intraclonal mutations') showed that the V(κ) of Toledo exhibited about 5-fold higher mutation frequency (MF) than the background level of Taq polymerase error (~0.12% mut/bp). Similarly, the MF of V(κ) of two of the BL cell lines was 3-4-fold higher than the Taq polymerase misincorporation rate. In contrast, the mutation frequencies of the V(κ) of DG-75, Farage and Pfeiffer did not significantly exceed the level of Taq polymerase error. Our combined results show that 5 out of the 6 B-cell lines studied originated from B-cells that have already somatically mutated in vivo their rearranged V(κ) genes. Moreover, two of the Burkitt's and one of the B-NHL cell lines exhibit intraclonal variation indicating that the process of somatic hypermutation continued following the neoplastic event, either in vivo or in culture. These results are in accord with the presumed origin of the majority of the BL and some types of the B- NHL, from centrocytes or centroblasts of the germinal centers in which the process of somatic hypermutation is taking place.