Determining the Mechanism of Transformation of Follicular Lymphoma into Diffuse Large B Cell Lymphoma.

The molecular mechanisms involved in histological transformation of follicular lymphoma (FL) to diffuse large B-cell lymphoma (DLBCL) are largely unknown. Here we investigated the clonal relationship in FL and DLBCL in 17 cases of paired FL/DLBCL samples and also in a bone marrow transplant (BMT) model. The aim of this study was to determine the cell of origin of the transformed DLBCL (t-DLBCL) and whether this cell always arises as a clonal evolution from the major FL clone. The immunoglobulin variable heavy chain (IgVH) was amplified by PCR using family specific primers, the major clone identified by homo/heteroduplex and genealogical trees generated. As expected 70% of FL/DLBCL cases used VH3, 24% VH4 and 6% VH5. The median degree of somatic hypermutation (SHM) was 15% (range 4 to 43%). In 3/17 cases the pattern of SHM was identical between FL and DLBCL. In 6/17 cases a clear pattern of direct evolution was evident. Surprisingly for 8/17 cases the pattern of SHM was compatible with the FL and DLBCL arising from a precursor cell rather than direct evolution. Clone specific primers and probes were designed to investigate whether there was evidence of the t-DLBCL in the earlier biopsy. Quantification by RQ-PCR demonstrated that in most cases the transformed clonotype was already present at levels between 10-4 and 10-2. We further examined an interesting case in which both the father (donor) and the son (recipient) developed FL and t-DLBCL 3 and 10 years after transplantation respectively. Sequencing confirmed the presence of an identical t(14;18) translocation in donor and recipient and that both cases used IGHV3-48*03. Analysis of the genealogical trees of SHM in these cases was also consistent with the evolution of t-DLBCL from a common precursor cell rather than direct evolution. These data are consistent with two distinct patterns of transformation of FL to t-DLBCL. About 50% of our cases had evidence of direct evolution while in the remaining 50% the pattern is more suggestive of the FL and t-DLBCL arising from a common precursor cell. The long latency period after BMT is in agreement with the requirement for additional genetic events within the t(14;18) bearing post germinal centre precursor lymphoma cell for the development of these diseases. The finding of the t-DLBCL clonotype in the FL sample before transformation suggests that the transforming event may occur in a minor subclone of the disease or earlier in a lymphoma precursor cell. Ongoing experiments seek to investigate the genetic events arising in these cells that result in transformation.