Aid-Mediated Hypermutation of B-Cell Receptors with Subsequent Subclone Selection Define Biological Differences Between IgM-Expressing and Isotype-Switched Follicular Lymphoma

Abstract 680

Pathogenesis of follicular lymphoma (FL) progresses from acquisition of the t(14;18) with upregulation of the anti-apoptotic BCL2 protein to establishment of neoplastic follicles after entry of FL cells into germinal centers (GC), and eventual transformation to diffuse large B-cell lymphoma. The initial t(14;18) appears to occur during immunoglobulin (Ig) VDJ recombination in the bone marrow. FL cells in GC persistently express activation-induced cytidine deaminase (AID) that continuously introduces mutations in the Ig variable region (IgV). Eventually, this somatic hypermutation (SHM) process generates novel glycosylation sites within the antigen-binding regions that are subsequently preserved. The rules governing subclone evolution upon entry into the GC until acquisition of glycosylation sites are unclear.

Based on an unbiased A-PCR amplification strategy, a mutation analysis of a total of 585 lymphoma-derived IgV sequences from 79 FL cases demonstrated a different mutation pattern in isotype-switched (IgG/IgA-FL; n=34) in comparison to non-switched (IgM-FL; n=45) FL. Despite lack of evidence for differences in the total numbers of mutations [IgM-FL: median 29.5 (range 1–182) vs. IgG/IgA-FL: 34 (12–121); p=0.15], IgG/IgA-FL showed accumulation of silent mutations in the VDJ region [15 (4–30) vs. 11 (0–48); p=0.010] with a lower replacement-to-silent (R/S) mutation ratio in complementarity determining regions (CDR) [1.8 (0.5–6) vs. 2.6 (0–13); p=0.024]. Most of IgG/IgA-FL showed evidence of selection against replacement mutations in CDR as determined by 3 different independent algorithms (Chang: 61%; Lossos: 32%; Hershberg: 78%), arguing for active preservation of a selected BCR with a given antigen binding site. In contrast, a significant number of IgM-FL (Fisher's exact test: p=0.002) was characterized by an unambiguous footprint of positive selection as seen in normal B cells during affinity maturation. FL displaying evidence for BCR preservation according to these algorithms also showed a longer diversification process of the malignant clones compared to cases with active subclone selection as determined by a higher outgoing degree and path length in clonal diversification analysis (2.7±0.1 vs.3.1±0.1; p=0.01).

In order to explore whether AID activity had an influence on the hypermutation pattern, AID expression was measured by quantitative PCR and immunohistochemistry in a subgroup of 16 IgG/IgA-FL and 23 IgM-FL with sufficient archived material. While AID levels did not differ overall between both groups, AID expression was positively correlated with somatic IgV mutations in IgM-FL (r=0.48; p=0.03). Surprisingly, AID expression tended to be inversely correlated with IgV mutations in IgG/IgA-FL (r= −0.5; p=0.07).

Our data indicate opposing possible outcomes of the selection pressures acting in FL: In some cases, AID-induced subclones are positively selected based on advantageous variations in antigen binding sites. In this FL subgroup, AID effects are directly dependent on its expression level, and isotype switching has not yet occurred. Shorter diversification paths suggest an earlier stage in FL development. Overall, this FL subtype resembles physiological clonal selection of normal B cells driven by improved affinity for the cognate antigen. In contrast, the second FL subtype appears to be dependent on a particular BCR that is preserved against continuous AID attack. Hence, AID levels no longer correlate with the mutation rate. Most cases of this FL group have undergone isotype switching, and longer diversification paths suggest a later maturation stage.

In summary, we provide evidence for a maturation model with FL evolution from an early stage with positive, possibly antigen-driven subclone selection to a later stage characterized by BCR preservation. Indeed, one informative case with two FL subpopulations expressing the same VDJ sequence, one as IgM and the other as IgG, supports this hypothesis through evidence of positive selection in the IgM compartment and BCR preservation in the isotype-switched compartment. Nevertheless, an alternative scenario with early dichotomy between both defined FL subtypes cannot be excluded with sufficient certainty by this study. The consequences of the differences between the identified biological FL subtypes with respect to overall prognosis and risk for histological transformation remain to be evaluated.