Widespread BCL6 Rearrangement with Novel Non-Fusogenic Partner Loci Including Common Fragile Sites in B-Cell Non-Hodgkin Lymphoma Cell Lines.

BCL6 (at 3q27) is highly expressed in mature B-cells within the germinal center where immunoglobulin (IG) genes undergo somatic hypermutation and class switch recombination. BCL6 is believed to downregulate apoptotic damage responses by p53 induced by physiological DNA breakage therein. In germinal-center-derived B-cell non-Hodgkin lymphomas (B-NHL), such as follicular lymphoma and diffuse large B-cell lymphoma, BCL6 may be deregulated by juxtaposition with IG loci, or by promoter exchange with a variety of other (usually) hematopoietic genes of which >20 have been described hitherto. FISH analysis of well characterized cell lines from patients with B-NHL revealed 9/20 (45%) with previously undetected, exclusively non-IG, BCL6 rearrangements. These included 6 cell lines with breakage at the canonical breakpoint cluster region (BCR), 5 of which involved new partner loci, at 1q42, 3q12, 7q32, 8q24, 12p11, the sixth (MBNL1 at 3q25) having been described once previously. Fine-mapping with tile-path fosmid clones revealed that translocations involving the BCL6 BCR with partner loci at 1q42, 3q12, 7q32, and 8q24 were unlikely to involve classical promoter exchange, as no strand-compatible partner genes or transcripts map to these breakpoints. This was tested both by RT-PCR using primers for BCL6 BCR together with all known compatible ESTs and in silico genes at the breakpoint loci (according to UCSC May 2004 assembly), and by 5′-RACE. Intriguingly, the majority of these partner breakpoints mapped to common fragile sites, including FRA1H (1q42), FRA7H (7q32), and FRA8C (8q24). Treatment of the t(3;7)(q27;q32) cell line with aphidicolin preferentially induced breakage within the FRA7H breakpoint on the non-rearranged homolog, implying a stabilizing effect for this translocation. This cell line thus affords a unique model for chromosome fragility contributory to hemopoietic neoplasia. The break at 8q24 was effected by formation of a non-reciprocal der(8)t(3;8)(q27;q24) which generated head-to-head juxtaposition of the BCL6 regulatory region with that of MYC which was over-expressed in the cell line involved. Similarly, 1/3 remaining B-NHL cell lines which evidenced BCL6 breakage outwith the BCR also involved juxtaposition with MYC which again was over-expressed. A further non-BCR B-NHL cell line exhibited hemizous BCL6 loss concomitant with gross somatic hypermutation of the residual upstream regulatory region and BCL6 overexpression. In summary, our data show: 1) an unexpected variety of novel BCL6 rearrangement at the apparent expense of IG locus juxtaposition; 2) a novel (third) category of BCL6 partnership involving the BCR region apparently together with non-coding DNA; 3) the likely involvement of BCL6 in MYC cis-regulation, and 4) the contribution of chromosomal fragility to lymphomagenic BCL6 rearrangement. Future studies will address the contribution of DNA stress-stability at BCL6 translocation breakpoints.