De Novo t(14;18) Diffuse Large B-Cell Lymphoma Is Characterized by a Germinal Center B Cell-Like Phenotype and Acquisition of Glycosylation Sites by Somatic Mutation in Immunoglobulin VH Genes.

The t(14;18) translocation is a hallmark of follicular lymphoma (FL) but has been reported to occur ~ in 20% of de novo (primary) diffuse large B-cell lymphomas (DLBCL). To determine the cell of origin in de novo t(14;18)+ DLBCL, both phenotype, defined by germinal center B cell-like (GCB) / non-GCB criteria, and Ig VH gene usage were determined in 9 previously untreated primary cases. Each of the cases was carefully reviewed and selected by the absence of any prior history of FL. Cases with additional translocations detected by conventional cytogenetic analysis involving BCL-6 (3q27) or c-MYC (8q24) were excluded. In the 9 selected cases, there was no evidence for follicular structures identifiable by morphology or by immunostaining for follicular dendritic cells (CNA42 and CD23), and all cases were classified as centroblastic. Immunohistochemical data revealed expression of BCL-2 and BCL-6 in 9/9 (100%) cases. Immunostaining for CD10, BCL-6 and MUM1 defined 8/9 cases (88%) as GCB and 1/9 as non-GCB. Of these, 4/9 cases (44%) co-expressed BCL-6 and MUM-1, indicating that the expression of the two proteins is not mutually exclusive. The proliferation index assessed by Ki-67 expression was > 50% in 7/9 cases. BCL-2, BCL-6, AID and c-MYC genes expression were further assessed by real time PCR assays in the 9 t(14;18) + DLBCL cases and compared with 14 cases of typical t(14;18)+ FL. No significant difference was observed in the mean levels of expression of BCL-2 (0.64, range 0.12 to 2.11), BCL-6 (1.03, range 0.05 to 4.08), c-MYC (1.96, range 0.77 to 6) or AID (0.76, range 0.03 to 3.22) between the two lymphoma cohorts. VH gene usage was identified in 9 t(14; 18) + DLBCL cases by RT-PCR assays using a mix of VH leader and JH primers, with the amplified DNA cloned for analysis. VH3 usage was common (3 VH3-23, 2 VH3-72, 2 VH3-48, 2 VH3-07, 1VH3-74). In 9/9 cases, VH genes were invariably somatically mutated, and all were potentially functional. The average mutational load was high and ranged from 8.4 to 24.4% deviation in homology to germline. Evidence for a low level of ongoing somatic mutation, observed as intraclonal heterogeneity, was apparent in 2/9 cases (7 to 10 clones analysed/case). Strikingly, somatic mutations yielded potential N-glycosylation sites identified as Asn-X-Ser/Thr motifs in 7/9 (78%) tumor-derived VH genes, a frequency significantly elevated above levels reported both in normal B cells (9%) and in randomly analysed DLBCL cases (44%), but directly comparable to the frequency of acquired glycosylation sites described in t(14;18) FL (79%). Of the acquired glycosylation motifs in t(14;18)+ DLBCL here, 6/7 (86%) were located in the CDR3 domain, suggestive of positive selection. No correlation was observed between VH mutational load and BCL-6, AID, c-MYC or BCL-2 gene expression. To conclude, the predominant GCB phenotype and, more importantly, generation of novel glycosylation sites in VH genes by somatic mutations provide compelling evidence for a common cell of origin linking evolution of de novo t(14;18)+ DLBCL and t (14;18)+ FL.