No abstract

Follicular lymphoma (FL) is initiated by the t(14:18) that places the anti-apoptotic proto-oncogene BCL2 under transcriptional control of the immunoglobulin heavy chain (IgH) locus during primary VDJ recombination. Neoplastic FL B cells are arrested at the germinal center stage with ongoing somatic hypermutation of B-cell receptor (BCR) genes by activation-induced deaminase (AID). Antigen recognition by the clonal BCR may facilitate malignant growth upon entry of t(14;18)-immortalized naïve B cells into secondary lymphoid organs. We have reported at ASH 2012 that FL may be classified according to the AID-mediated BCR evolution pattern into two distinct subgroups: A predominantly IgM-expressing subgroup with evidence for antigen-driven affinity maturation, and a subgroup selected for BCR sequence preservation that has often undergone class switch recombination. To investigate whether these BCR selection patterns were correlated to clinical outcome, we retrospectively analyzed somatic hypermutation of multiple BCR sequences in 66 patients (median age: 49; range: 29-75 years) with advanced-stage FL that had undergone a research tumor rebiopsy after inclusion into our prospective idiotype vaccination program (Blood, 2011) with informed consent. Biopsies were performed at a median interval of 20.1 months from diagnosis (range: 0-171 months). At rebiopsy, 43 patients had not yet received systemic cytoreductive therapy (chemotherapy, antibody therapy, or radiation therapy); 14 and 9 patients had been treated with one or two prior regimens, respectively. IgH transcripts were cloned with an unbiased anchored PCR strategy with nested constant region-specific primers. A median of 8 clonal IgH sequences was subjected to bioinformatic analysis for BCR selection patterns by the focused test (Hershberg et al., 2008). 22 patients (33.3%) were classified as having evidence (focused test: p>0) for positive BCR selection through antigen-driven affinity maturation; 44 patients (66.6%) belonged to the BCR preservation category defined by p<0. Clinical risk according to the FLIPI was evenly distributed among both groups (BCR selection: 5 cases low FLIPI, 15 intermediate, 2 high; BCR preservation: 15, 22, 7). The BCR category distribution of app. 1:2 did not change with increasing time from diagnosis to biopsy or after prior therapy. Among 39 patients who were managed initially with a “watch and wait” policy (36 of these underwent rebiopsy prior to initiation of therapy), BCR selection was associated with superior progression-free survival with a median of 87.7 months versus 16.9 months in the BCR preservation group (log-rank test: p=0.024). The treatment-free interval during which the rebiopsy was taken was longer in the BCR selection category for all patients (median 102.0 versus 31.0 months; p=0.030) and for patients with initial “watch and wait” policy (median 103.4 versus 21.0 months; p=0.049). Transformations to aggressive lymphoma occurred exclusively in the BCR preservation group (n=4). At a median total follow-up from diagnosis of 101.6 months, 8 patients (36,4%) in the BCR selection group and 7 patients (15,9%) of the BCR preservation group have not yet received any cytoreductive therapy. When death or transformation were defined as competing events, median event-free survival was not reached in either group with 2 events in the BCR selection group and 8 events in the BCR preservation group (p=0.10). The BCR selection pattern as defined by the focused test through analysis of multiple BCR sequences may represent a novel prognostic factor for the natural history of FL in the pre-treatment phase. A focused test-based categorization could be readily incorporated into the diagnostic work-up of FL and could potentially complement clinical prognostication by means of the FLIPI score. Since the retrospective design of this study cannot entirely exclude possible selection bias, the hypothesis that antigen-driven BCR affinity maturation defines a favorable FL subgroup calls for validation in a prospective study. The relationship of the BCR selection pattern with the established prognostic role of the tumor microenvironment warrants investigation. In summary, we propose that BCR selection may govern the natural history of follicular lymphoma in treatment-free periods, thereby lending further support to the concept that BCR signaling plays an important causal role in FL lymphomagenesis. Disclosures: No relevant conflicts of interest to declare.

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. Disclosures: No relevant conflicts of interest to declare.