The Pattern of Immunoglobulin Variable Genes Indicates That Most Post-Transplant Lymphoproliferative Disorders Derive from B-Cells That Have Failed the Germinal Center Reaction.
Authors: Capello Daniela , Cerri Michaela , Berra Eva , Rossi Davide , Morra Enrica , Muti Giuliana , Paulli Marco , Gaidano Gianluca 
Published: October 2019

Abstract
Monoclonal posttransplant lymphoproliferative disorders (PTLD) comprise polymorphic PTLD (P-PTLD), diffuse large B cell lymphoma (DLBCL) and Burkitt/Burkitt-like lymphoma (BL/BLL). Recent studies have elucidated the germinal center-origin (GC) of PTLD, yet a detailed analysis of IgVH and IgVL chain genes is lacking. We investigated 54 PTLD, including 16 P-PTLD, 35 DLBCL and 3 BL/BLL for usage, mutation frequency and mutation pattern of clonal IgVH and IgVL rearrangements. A functional IgVH rearrangement was identified in 47/54 (87.0%) cases. Four cases yeilded only an out of frame IgVH rearrangement or a rearrangement rendered nonfunctional by crippling mutations. Three cases showed hybrid Ig VDJ rearrangements: two cases with a V-V fusion rearrangement and one case with a J-J fusion rearrangement, suggesting a failed attempt of heavy chain receptor revision in GC reaction. Despite extensive investigation by multiple PCR strategies, a functional IgVL rearrangement was found in only 25/54 (46.3%) cases. Eleven out of 25 (44.0%) cases harbored IgV kappa rearrangements and 12/25 (48.0%) cases harbored functional IgV lambda rearrangements. Two cases showed the presence of both IgV kappa and IgV lambda functional rearrangements. Among PTLD carrying solely nonfunctional IgVL rearrangements, 7/54 (13.0%) cases showed a crippled rearrangement and 11/54 (20.4%) cases harbored only an out of frame and/or inactivated IgV kappa gene. Inactivation occurred by rearrangement involving the kappa-deleting element (KDE). In 11/54 (20.4%) cases, no IgVL rearrangement was identified. Overall, only 23/54 (42.6%) PTLD displayed both a functional IgVH and a functional IgVL rearrangement. Analysis of somatic hypermutation showed the presence of somatically hypermutated IgVH and/or IgVL genes in 45/54 PTLD (83.3%). Conversely, IgV rearrangements of 9/54 (16.6%) PTLD were in germline configuration, suggesting a derivation from B-cells that have not experienced the GC-reaction. Among mutated cases, the average mutation frequency was 8.83% (median 8.43%, range 2.10%–24.1%) for IgVH genes and 7.37% (median 6.71%, range 2.30%–26.0%) for IgVL genes. Thirty-two cases (71.1%) showed highly mutated (mutation frequency >6%) IgVH and/or IgVL genes, a condition that, in normal B-cell, results in lower affinity for antigen and apoptosis. Analysis of the distribution of replacement and silent mutations in functional IgVH and/or IgVL sequences showed tendency to conserve FR sequences and maintain antigen binding in 20/34 (58.8%) cases. Selection for high affinity antigen binding occurred in 14/34 (41.2%) cases. Our data suggest that most PTLD arise from B-cells that have experienced the GC-reaction and frequently display impaired B-cell receptors (BCR). Since a functional receptor is required for normal B-cell survival during GC transit, PTLD development may implicate rescue from apoptosis and expansion of B-cells that have failed the GC-reaction. Notably, virtually all PTLD with nonfunctional IgVH and/or IgVL rearrangements carried EBV infection, which may promote cell survival.