Follicular Lymphoma Development Research Articles

Analysis of immunoglobulin/T-cell receptor repertoires by high-throughput RNA sequencing reveals a continuous dynamic of positive clonal selection in follicular lymphoma.

Follicular lymphoma (FL) course is highly variable, making its clinical management challenging. In this incurable and recurring pathology, the interval between relapses tends to decrease while aggressiveness increases, sometimes resulting in the transformation to higher-grade lymphoma. These evolutions are particularly difficult to anticipate, resulting from complex clonal evolutions where multiple subclones compete and thrive due to their capacity to proliferate and resist therapies. Here, to apprehend further these processes, we used a high-throughput RNA sequencing approach to address simultaneously the B-cell immunoglobulin repertoires and T-cell immunoglobulin repertoires repertoires of lymphoma cells and their lymphoid microenvironment in a large cohort of 131 FL1/2-3A patients. Our data confirm the existence of a high degree of intra-clonal heterogeneity in this pathology, resulting from ongoing somatic hyper-mutation and class switch recombination. Through the evaluation of the Simpson ecological-diversity index, we show that the contribution of the cancerous cells increases during the course of the disease to the detriment of the reactive compartment, a phenomenon accompanied by a concomitant decrease in the diversity of the tumoral population. Clonal evolution in FL thus contrasts with many tumors, where clonal heterogeneity steadily increases over time and participates in treatment evasion. In this pathology, the selection of lymphoma subclones with proliferative advantages progressively outweighs clonal diversification, ultimately leading in extreme cases to transformation to high-grade lymphoma resulting from the rapid emergence of homogeneous subpopulations.

Emergence of Highly-Plastic B Cell States Cooperates with Early Immune Microenvironment Remodeling to Drive Follicular Lymphomagenesis

Follicular lymphoma (FL) is a prototypical example of B cell lymphoma arising from germinal center (GC) B cells. Recent single cell transcriptomic studies investigating the transcriptional heterogeneity of FL suggest that tumor B cells are functionally diverse with cells acquiring an intermediate phenotype between GC and memory (mem), in line with the premalignant FL ontogeny where BCL2 + memory B cells require multiple re-entries into the GC to facilitate the transformation process. Since FL cells often hijack the epigenetic machinery through mutations in histone modifying proteins - notably KMT2D - we questioned here how epigenetic alterations contribute to the emergence of this intratumoral heterogeneity. We developed a genetically engineered mouse FL-like model (GEMM) carrying Kmt2d loss-of-function in B cells and BCL2 overexpression recapitulating different progression stages from preneoplastic hyperplasia to FL-like tumors. We analyzed 45,541 high-quality single cell transcriptomes and immune repertoires (B cell receptor, BCR and T cell receptor, TCR) from 18 mice including immunized-control mice in the context of physiological GC reaction. We developed a computational workflow on mutant B cells combining supervised and unsupervised metaclustering approaches in order to highlight recurrent gene programs active in preneoplastic hyperplasia and the various stages leading to FL-like tumors. We used inferCNV to assess the association between genetics and transcriptomic heterogeneity and inferred cell-cell interactions to characterize the microenvironmental determinants associated with B cell heterogeneity throughout FL progression. By modeling the GC cycle dynamics and building B cell signatures during a normal immune response, we found that our FL GEMM mirrored the desynchronization of the GC reaction program previously observed in human FL. Single cell expression profiles of Kmt2d-BCL2 mutant cells also showed a reproducible increase in phenotypic heterogeneity during FL progression, manifesting as a continuum of cell states from proliferating GC-like to quiescent mem-like cell states. We defined this GC-to-Mem axis as the major source of intra-tumor transcriptional heterogeneity. Single-cell BCR sequencing revealed the emergence of highly-plastic B cell states during FL evolution, co-existing within individual mice and being largely independent of subclonal genetic variation. To further elucidate the emergence and maintenance of these cell state transitions, we explored the phenotypic and transcriptional changes within the immune microenvironment along disease progression. Significant compositional changes were found, including the expansion of T follicular helper cells and T regulatory T cells together with the loss of naive CD4, CD8 T, and NK cell populations, preceding malignant transformation and creating a (pre)tumoral supportive niche persisting in advanced tumors. Prior remodeling of a premalignant supportive immune niche may be crucial in establishing malignant cell heterogeneity. Ongoing efforts aim to impair tumor-immune interactions driving tumor B cell dynamics and survival. In conclusion, our results provide a high-resolution view of events spanning FL progression and indicate a major role of early TME remodeling in establishing a suitable niche for progression. Early intervention aimed to target the B cell-tumor microenvironment interactions driving intra-tumoral heterogeneity may represent a promising therapeutic avenue against early disease and prevention of FL recurrence.

Follicular lymphoma evolves with a surmountable dependency on acquired glycosylation motifs in the B-cell receptor

AbstractAn early event in the genesis of follicular lymphoma (FL) is the acquisition of new glycosylation motifs in the B-cell receptor (BCR) due to gene rearrangement and/or somatic hypermutation. These N-linked glycosylation motifs (N-motifs) contain mannose-terminated glycans and can interact with lectins in the tumor microenvironment, activating the tumor BCR pathway. N-motifs are stable during FL evolution, suggesting that FL tumor cells are dependent on them for their survival. Here, we investigated the dynamics and potential impact of N-motif prevalence in FL at the single-cell level across distinct tumor sites and over time in 17 patients. Although most patients had acquired at least 1 N-motif as an early event, we also found (1) cases without N-motifs in the heavy or light chains at any tumor site or time point and (2) cases with discordant N-motif patterns across different tumor sites. Inferring phylogenetic trees of the patients with discordant patterns, we observed that both N-motif–positive and N-motif–negative tumor subclones could be selected and expanded during tumor evolution. Comparing N-motif–positive with N-motif–negative tumor cells within a patient revealed higher expression of genes involved in the BCR pathway and inflammatory response, whereas tumor cells without N-motifs had higher activity of pathways involved in energy metabolism. In conclusion, although acquired N-motifs likely support FL pathogenesis through antigen-independent BCR signaling in most patients with FL, N-motif–negative tumor cells can also be selected and expanded and may depend more heavily on altered metabolism for competitive survival.

Prevalence of Acquired N-Glycosylation Sites at the Single Cell Level in Follicular Lymphoma

Follicular lymphoma (FL), a malignancy derived from germinal center B cells, is characterized by ongoing somatic hypermutation of the immunoglobulin variable region (IGV) genes. This somatic hypermutation can introduce novel N-glycosylation sites (N-sites), consisting of Asn-X-Ser/Thr motifs, in the IGV resulting in the addition of glycans at these sites. Binding of lectins, such as DC-SIGN, to these glycosylated surface immunoglobulins (i.e., B cell receptors) activates the B cell receptor pathway in an antigen-independent manner, promoting FL B cell survival and proliferation. Previous studies have concluded that the acquisition of N-sites is a stable and clonal event during FL evolution (Odabashian et al., Blood 2020). Here, we investigated the dynamics and potential impact of N-site prevalence at a single cell level, in paired heavy and light chains, across sites and over time in 16 patients with FL. We profiled tumor fine needle aspirates from patients enrolled in two clinical trials (NCT02927964, NCT03410901). 15 of 16 patients were previously, but not recently treated, while one was previously untreated. Samples were collected from different tumor sites before treatment, on-treatment, and at tumor progression. Single cell suspensions were subjected to single cell B cell receptor (scBCR) and RNA (scRNA) sequencing using the 10X chromium platform. Only BCR sequences belonging to patient-specific dominant malignant heavy and light chain clones were considered for further analysis. In total, we analyzed more than 250,000 FL tumor cells from 89 tumor samples from 16 FL patients. While most patients acquired at least one N-site as an early and stable event that was conserved across distinct tumor sites and over time, 3 patient cases proved to be exceptions: one patient had no detectable N-sites in either the heavy or light chain IGV genes at any tumor site or timepoint. In a second patient, only one of two tumor sites exhibited a novel N-site, and this was probably acquired after divergence of the tumor populations to distinct tumor sites (Haebe, Shree et al., Blood 2021). Interestingly, clinical tumor progression in this patient arose from selection and expansion of an N-site negative subclone, despite the putative survival advantage conferred by acquired N-sites. In the third patient, N-sites were acquired in a precursor cell before subclonal divergence, but subsequently overtaken by an N-site-negative subpopulation, which comprised up to 90% of tumor cells at a given site (Figure 1A). These results suggest that other acquired features can out-compete the selective advantage of N-glycosylation. To elucidate biological differences between cells with and without N-sites within each sample, we integrated scBCR and scRNA data for the third patient using shared cell barcodes. N-site positive cells exhibited a higher expression of the BCR pathway and inflammatory response, while cells without N-sites had higher activity of metabolic processes, including oxidative phosphorylation and glycolysis (Figure 1B). In conclusion, while acquired N-sites likely support FL pathogenesis through antigen-independent BCR signaling in many FL patients, selection and expansion of N-site negative cells in some patients in our study suggests that N-site negative cells can thrive, and likely depend on other pathways, with our preliminary results pointing to differential metabolic regulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evolution in the Definition of Follicular Lymphoma and Diffuse Large B-Cell Lymphoma: A Model for the Future of Personalized Medicine

The definitions of follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are evolving in the era of personalized medicine. Early stages of the evolution of FL have been recognized. Two histological manifestations of early lesions are in situ follicular neoplasia and duodenal type FL. Additionally, FL frequently undergoes histological transformation, the most common form being DLBCL. High-grade B-cell lymphoma with double hit, with translocations involving BCL2 and MYC are important clinically. Rarer forms of transformation include classic Hodgkin lymphoma (CHL) and histiocytic sarcoma. In addition to conventional FL associated with the BCL2 translocation, alternative forms of BCL2-negative FL have been observed. These are heterogenous clinically and genetically. A distinctive group of B-cell lymphomas of follicle cell derivation arise in young patients and include pediatric type FL, testicular FL and a large B-cell lymphoma with IRF4 rearrangement. Historically DLBCL was separated into only two histological variants, centroblastic and immunoblastic. In 2017 the WHO classification recommended (1) the segregation of activated B cell and germinal center B cell derived DLBCL, (2) the identification of high-grade B-cell lymphoma with double hit, and (3) the recognition of an aggressive lymphoma that may resemble Burkitt lymphoma, currently designated in the International Consensus Classification as Large B-cell lymphoma with 11q aberration. Today we appreciate greater genomic complexity among aggressive B-cell lymphomas. Recent studies with NGS and mutational profiling have identified clinically significant genetic subgroups. It is hoped that these data ultimately will lead to targeted therapy based on the genetic profile.

Regional Differences in Lymphomagenesis Identified through Quantification of AICDA-Mediated Subclonal Evolution in Follicular Lymphoma: Do Pesticides Play a Role?

Background:Environmental risk factors for lymphoma are ill-defined and controversial. Epidemiologic studies have demonstrated an association between pesticide exposure and an increase in non-Hodgkin lymphomas (NHL), particularly those with a 14;18 translocation. However, in the absence of a mechanistic explanation for the effects of pesticide on lymphomagenesis, the data remain correlative and subject to alternative explanations.Mutagenesis due to activation-induced cytidine deaminase (AICDA) is key to lymphomagenesis, particularly in follicular lymphoma (FL). AICDA exerts physiologic effects by creating mutations in the immunoglobulin heavy locus (IGH) for somatic hypermutation in order to produce highly specific antibodies. AICDA's off-target genomic damage creates “driver” and “passenger” mutations, which can be used to quantify the subclonal evolution of each tumor specimen. Using deep sequencing (10000-fold) with appropriate error-thresholding, our lab previously demonstrated that the number of AICDA-mediated mutations in the 5' UTR of BCL2 is linearly related to the sum of AICDA-mediated mutations at other off-target sites, implying that mutations within the 5'UTR of BCL2 can function as a surrogate marker for the genome-wide aberrant targeting (Spence, J Immunology, 2014). We have also demonstrated that the number of mutations within IGHV is unrelated to the sum of genome-wide aberrant mutations. The sequencing data can thus be used to determine the minimum number of subclonal populations within a specimen as defined by each region (BCL2 and IGHV).We hypothesized that the number of AICDA-generated subclones would differ in patients with FL living in high and low pesticide use regions, elucidating whether pesticide-associated lymphomagenesis acts through AICDA or an alternative mechanism. We sought to assess the feasibility of demonstrating a difference in extent of AICDA-mediated subclonal evolution in patients with FL residing in regions with high and low pesticide usage by quantifying AICDA-mediated subclones in the 5' UTR of BCL2 and within IGHV.Methods:Using a state-wide database of pesticide application organized by ZIP code, we identified 35 patients with FL seen at our institution in Western New York in locales with high (N=19) and low (N=16) pesticide usage and quantified AICDA-mediated subclones in the 5' UTR of BCL2 and within IGHV for these patients' FL specimens. This sample size provided 75-92% power to detect a difference in the number of subclones by pesticide usage, using a 2-sided 0.05 level Wilcoxon test, assuming an effect size of 75-80% probability of more subclones in one pesticide usage group.Results:We identified 19 patients from high pesticide use (1000+ gigaliters (GL)) and 16 from low pesticide use ZIP codes (<1000 GL). Pesticide volume use by ZIP code in the low group ranged from 10-664 GL with a median of 263 GL, and in the high group ranged from 1636 to 67,140 GL with a median of 4787 GL.Patients living in regions of high and low pesticide use were similar in terms of race and ethnicity. The proportion of women was also similar: 43% among high versus 50% among low. Mean age at diagnosis was 62 in the high versus 60 in the low group. Three patients were diagnosed at age < 45 in the high group while no one in the low group was diagnosed at age < 45.BCL2 subclone count was lower (p = 0.046) among those in high pesticide ZIP codes (median 3.0), compared with those in low pesticide ZIP codes (median 6.5). Although the median IGH subclone count was 278 among those in the high group versus 91.5 in the low group, there was insufficient evidence of an association between IGH subclone count and the level of pesticide usage (p = 0.36).Conclusions:The extent of subclonal evolution was significantly different between lymphoma specimens of patients residing in ZIP codes with high versus low pesticide usage. Our data suggest that AICDA-mediated subclonal evolution is negatively correlated with residence in a ZIP code with high pesticide use, raising the question of whether pesticides could induce lymphoma through an alternate mechanism. This observation calls for both corroboration and examination of alternative hypotheses. We demonstrate a feasible approach to assessing these questions in a larger dataset. DisclosuresPeterson: Abbott: Consultancy. Casulo: Verastem: Research Funding; BMS: Research Funding; Genentech: Research Funding; Gilead: Research Funding.

A Prospective Study of Clonal Evolution in Follicular Lymphoma: Circulating Tumor DNA Correlates with Overall Tumor Burden and Fluctuates over Time without Therapy

Background: Follicular lymphoma (FL) shows marked variation in clinical course including spontaneous regression and histologic transformation (HT). Watchful waiting (W&W) is routinely applied to pts with newly diagnosed FL, but monitoring strategies are not standardized. Pts with progression within 1-2y of diagnosis have worse outcomes, but the biologic basis is unclear and biologic-based classifiers are not routinely applied at diagnosis. Circulating tumor DNA (ctDNA) is a highly tumor-specific biomarker that is prognostic in aggressive B-cell lymphomas, but its ability to serially monitor FL remains undefined. We applied a next-generation sequencing assay to identify tumor clonotypes for serial monitoring of peripheral blood in pts with untreated FL as part of an ongoing prospective clinical trial [NCT03190928].Methods: Pts with grade I-II or 3A FL are eligible if evaluable disease on CT or FDG-PET, age ≥18, ECOG ≤2, no evidence of HT, and no prior systemic therapy. Pts undergo W&W until they meet uniform protocol-defined treatment criteria and remain on study until second-line therapy. Baseline testing includes labs, peripheral blood flow cytometry, BM biopsy/aspirate, CT and FDG-PET scans, and research biopsy. Pt have clinic visits every 4m for 2y, every 6m in years 3-5, then annually. CT scans are every 8m for 2y, then annually. FDG-PET scans are at baseline, at 2y, and any time of suspected progression. Peripheral blood samples including Streck tubes (plasma) and PBMCs are drawn at each clinic visit and stored. For ctDNA analysis, tumor DNA was amplified from FFPE using locus-specific primer sets for the Ig heavy-chain and light-chain loci along with BCL1/BCL2 translocations (Adaptive Biotechnologies). Amplified products were sequenced and tumor clonotypes were identified in plasma and PBMCs. Serial tracking of ctDNA was done in plasma and blinded to clinical outcomes.Results: 77 pts enrolled between July 2017 and July 2021. Median age was 57 (range 24-83) including 14 (18%) low-risk, 29 (38%) intermediate-risk, and 34 (44%) high-risk by FLIPI. Fourteen (18%) pts had stage I-II disease. Forty-three (56%) pts had monoclonal B-cells on peripheral blood flow cytometry. Twenty-nine (38%) pts progressed requiring frontline therapy including 7 (9%) pts with HT. Twenty-five (32%) pts were monitored ≥2y with no progression including 10 (13%) pts with evidence of at least some spontaneous regression by CT. Twenty (26%) pts were on study for <2y and 3 (4%) pts were unevaluable. Of 36 pts with FFPE analyzed, 35 (97%) had ≥1 dominant tumor clonotype identified for tracking. Of these, 32 (91%) had ≥1 dominant clonotype identified in plasma suitable for tracking. Three pts without available FFPE had the dominant clonotype identified from plasma that was used for tracking. In 28 pts, plasma was compared to PBMCs, and ≥1 clonotype was identified in both compartments in 21 (75%), plasma only in 5 (18%), and neither in 2 (7%). The median overall levels of baseline plasma ctDNA was 104.7 counts/mL (range 4-2096); progressors had median levels of 136.0 counts/mL (range 4-1493) compared to 40.9 count/mL (32-2096)(p=0.85) for non-progressors (Figure 1A). Baseline ctDNA correlated with the total metabolic tumor volume (TMTV) on FDG-PET scan (r=0.49, p=<0.006)(Figure 1B). Pts who required immediate treatment often had very high baseline levels of ctDNA (black dots, Figure 1C) and serial monitoring prior to progression demonstrated patterns of sharp increases in ctDNA prior to progression or relatively stable levels in others (Figure 1C). Notably, significant fluctuations were noted in ctDNA for non-progressing pts with an overall trend for decreasing values including four pts with evidence of spontaneous regression by CT with corresponding decreases in ctDNA over time (Figure 1D). As a notable examples, FL_14 had baseline ctDNA level of 2096 counts/mL with decreases to 101 counts/mL after 4 months, 14 counts/mL at 16 months, and no detectable ctDNA at 2 years.Conclusions: ctDNA quantified from plasma in FL mirrors TMTV. Serial monitoring of ctDNA in patients without therapy demonstrated various patterns of fluctuation, including some patients in which ctDNA became undetectable coincident with spontaneous clinical regressions. ctDNA thus provides a non-invasive platform to monitor the natural history of FL, enabling future studies of tumor immune surveillance in this disease. [Display omitted] DisclosuresJacob: Adaptive Biotechnologies: Current Employment, Current equity holder in publicly-traded company. Bagaev: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Meerson: BostonGene: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Postovalova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Kudryashova: BostonGene: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Kotlov: BostonGene Corp: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Fowler: BostonGene: Current Employment, Current holder of stock options in a privately-held company.

A Multicenter, Single-Arm, Phase I/II Dose Finding and Efficacy Study of Venetoclax, CC-486, and Obinutuzumab in Minimally-Pretreated Follicular Lymphoma

KC and ML are co-first authors.Background:Follicular lymphoma (FL) is the most common indolent B-cell lymphoma and has a relapsing and remitting course with risk of transformation to aggressive disease. Long-term toxicity can accumulate with repeated exposure to palliative cytotoxic chemotherapy. Rationally targeted agents have demonstrated disease control with limited toxicity in hematologic malignancies, representing a potential chemotherapy-sparing option.BCL2 rearrangements leading to overexpression are nearly universal in FL, but BCL2 inhibition monotherapy has disappointing efficacy in FL. Potential reasons include known resistance mechanisms, such as MCL-1 overexpression, or redundant survival pathways. Founder mutations in FL often involve epigenetic regulation, and BCL2 rearrangements are necessary but not sufficient for FL lymphomagenesis. In patients with FL, abnormal DNA methylation programming cooperates with somatic mutations to drive lymphomagenesis. The resulting epigenetic deregulation may be targetable by hypomethylating agents, including azacitadine (AZA). There are no published studies of venetoclax and AZA in preclinical models of FL, but there are extensive preclinical data in acute myeloid leukemia. AZA is an epigenetic modulator with synergistic anti-leukemic effect when paired with venetoclax. Treatment with AZA results in MCL-1 downregulation, which may abrogate FL resistance to BCL2 inhibition. AZA also induces re-expression of CD20 and re-sensitizes FL patients to anti-CD20 therapy. In lymphoma xenograft models, the combination of obinutuzumab and venetoclax causes more tumor growth inhibition compared to rituximab with venetoclax, possibly from more potent direct cytotoxicity.These preclinical studies suggest that epigenetic modulation of FL cells with AZA may increase the sensitivity to BCL2 inhibition and anti-CD20 therapy. The foundational position of epigenetic dysregulation in the clonal evolution of FL suggests the optimal time for this strategy may be early in the course of the disease. We hypothesize that a safe and tolerable dose of venetoclax, CC-486 (oral AZA), and obinutuzumab can be found with treatment efficacy equal to or better than other non-chemotherapy agents in the frontline treatment of FL. We present a dose-finding and efficacy trial of venetoclax, CC-486, and obinutuzumab in minimally pre-treated FL patients.Study Design:This is a single arm, multi-center phase I/II clinical trial (Figure 1). The dose finding phase is a 3+3 design with 3 escalating dose levels and two optional de-escalation levels (12-18 patients). Venetoclax (400-800mg) will be given days 1-28 of each 28 day cycle; CC-486 (150-200mg) will be given days 1-14; and obinutuzumab will be given at a fixed dose of 1000mg on days 1, 8, and 15 of cycle 1 and then day 1 of each subsequent cycle. Venetoclax and CC-486 will be given up to 12 cycles and obinutuzumab for a total of 9 cycles. Once the recommended phase 2 dose (RP2D) is identified, the study will proceed to a Simon two-stage expansion cohort. In this cohort, a three month “window” of the doublet venetoclax and CC-486 prior to introduction of obinutuzumab at cycle 4 will allow an assessment of activity of the two-drug combination. Obinutuzumab is given on days 1, 8, and 15 of cycle 4, and day 1 of each subsequent cycle. Key inclusion criteria are: age ≥ 18, ECOG ≤2; adequate kidney/liver function; and grade 1-3a FL with an indication for treatment who are either untreated or have ≤2 courses (16 doses) of lifetime anti-CD20 therapy. Patients with clinical or histologic evidence of FL transformation are excluded. Up to 32 patients will be enrolled to test the null hypothesis of a 30% CR rate against a 50% alternative (80% power, one-sided alpha=0.10).The primary objectives are the RP2D and safety based on adverse events for the dose-finding cohort, and the CR rate by PET in the expansion cohort. Secondary objectives include progression free survival, overall survival, effect of 3 cycles of venetoclax/CC-486 in the window design of the expansion cohort, and CR rate at 30 months post-treatment with PET. Exploratory objectives include pre-treatment mutation biomarkers, minimal residual disease by circulating tumor DNA, and circulating cell-free DNA methylation patterns. This trial design was reviewed and revised at ASH CRTI. Recruitment is ongoing and this trial is registered with ClinicalTrials.gov: NCT04722601. [Display omitted] DisclosuresKline: Kite/Gilead: Speakers Bureau; Seagen: Membership on an entity's Board of Directors or advisory committees; Regeneron: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Research Funding; Verastem: Research Funding; SecuraBio: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy, Membership on an entity's Board of Directors or advisory committees. Smith: Celgene, Genetech, AbbVie: Consultancy; Alexion, AstraZeneca Rare Disease: Other: Study investigator.

IGHV sequencing reveals acquired N-glycosylation sites as a clonal and stable event during follicular lymphoma evolution

AbstractFollicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells' dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.

High-Throughput BCR Sequencing and Single-Cell Transcriptomics Reveal Distinct Transcriptional Profiles Associated with Subclonal Evolution of Follicular Lymphoma

Objectives: Follicular lymphoma (FL) typically originates from premalignant mature B cells that carry the founder t(14;18) BCL2 translocation. Mutations in epigenetic modifiers and acquisition of N-glycosylation sites in CDR regions of the B-cell receptor (BCR) are recurrent secondary events in FL pathogenesis. Despite these oncogenic drivers, FL can remain indolent and clinically stable for years. The molecular events driving subclonal evolution into symptomatic progression and eventual transformation to aggressive lymphoma are insufficiently understood. FL cells are frozen in their B-cell development at the germinal center stage and undergo continuous somatic hypermutation mediated by expression of activation-induced deaminase (AID). We aim to identify crucial drivers of subclonal FL evolution by high-throughput mapping at single-cell resolution. Methods: Viable FL cells were isolated and cryopreserved from 23 histologically or immunocytologically confirmed FL samples from 13 patients with informed consent. Full-length VDJ/VJ transcripts were isolated by unbiased template-switching ARTISAN PCR and massive parallel NGS sequencing on the PacBio platform. The clonal primordial FL BCR (pBCR) was reconstructed from unmutated IGV/IGJ sequences with the CDR3 of the least mutated BCR. Since the IgTree program was unable to process the obtained numbers of BCR sequences, we developed the WILLOW algorithm for analysis of BCR evolution based on the principle of maximum parsimony and on distance from the pBCR. Intraclonal BCR variability was quantified by Shannon's diversity index. 5' single cell transcriptomics and VDJ/VJ sequencing was performed on 2 pools of highly purified FL cells from 5 lymph node biopsies on the 10x Genomics platform. Data were deconvoluted based on expressed variants by the Single Cell Sample Matcher (SCSM) algorithm. Clustering based on gene expression profiles was performed by shared nearest neighbour (SNN) modularity optimization within the R Seurat package. Genes whose expression differed significantly (adjusted p&amp;lt;0.05) between clusters were assigned to gene ontology terms. Results: ARTISAN PCR/PacBio NGS yielded a median of 743 full-length VDJ and VJ sequences (range 62-12782) per BCR chain with expected high intraclonal diversity (median 200 subclones, range 15-3301). WILLOW revealed dominant FL subclones with a subclonal hierarchy wherein multiple routes converged to offspring nodes with identical additional mutations rather than tree-like branching (Figure). In serial samples of 4 patients, lymph node biopsies had only marginally higher subclonal diversity than blood or bone marrow samples (p=0,055; Wilcoxon's matched-pairs signed rank test). Overall BCR mutational burden increased over time in sequential biopsies. Two cases of histological FL transformation were dominated by a single subclone (65% and 80% of all VDJ/VJ sequences, respectively) that was rare in the preceding FL BCR network (0.2% and 1.8%). Pooled transcriptomics data from 6050-6500 cells were assigned to individual samples by SCSM and revealed up to seven transcriptional clusters per FL. In 9 of 10 FL, genes assigned to immune function strongly contributed to separation into one or more clusters. Single cell VDJ/VJ sequencing yielded combined heavy and light chain BCR sequences for a median of 502 FL cells per biopsy (range 22 - 1919) that permitted mapping of subclonal evolution by WILLOW based on complete BCR information. Transcriptome clusters were not distributed evenly throughout the WILLOW FL BCR networks but rather statistically associated with distinct major FL subclones. Vice versa, major FL subclones within the same biopsy were distinguished by particular gene expression profiles. Conclusions: WILLOW facilitates mapping of subclonal FL evolution based on high-throughput BCR sequencing. FL evolution proceeds in networks rather than tree-like branching, whereby acquisition of certain combinations of several BCR mutations can occur in parallel in different trajectories. Transcriptomic profiling of single FL cells identifies distinct clusters within a single biopsy. Mapping of these clusters to the FL cell position in the subclonal FL evolutionary network identifies putative mechanisms that are associated with subclonal progression. These mechanisms involve physiological B-cell signalling pathways. Figure Disclosures No relevant conflicts of interest to declare.

Deep Profiling of Genetic Aberrations and Clonal Evolution in Follicular Lymphoma

Holte: Novartis: Honoraria, Other: Advisory board.

Dynamical Modeling of Clonal Evolution in Paired Primary and Relapsed Follicular Lymphoma Predicts a Link Between Cell Migration and Evolutionary Heterogeneity

Background: Recently, data of paired primary and relapsed t(14;18) positive follicular lymphoma (FL) revealed a large heterogeneity in clonal evolution. In these studies, ongoing somatic hypermutation (SHM) in immunoglobulin heavy chain V gene (IGHV) rearrangements was used as a "flight recorder" of evolution providing phylogenetic trees. Pasqualucci et al. [Cell Rep. 2014;6(1):130-40] observed that FL transformation to DLBCL is associated with "divergent evolution", i.e. primary and relapse tumors arise from a common precursor. Loeffler et al. [Genomic and epigenomic co-evolution in follicular lymphomas. Leukemia. 2015;29(2):456-63] reported a broader evolutionary spectrum in relapsed FL, ranging from "divergent" to "sequential" (relapse sequences emerge out of primary clones) and "no evolution" (shared IGHV sequences in primary and relapse tumors).Currently, there is no conclusive concept to explain these different types of evolution. Furthermore, the consequences of this heterogeneity for clinical decision-making are yet unclear. To answer these questions, a mathematical modeling approach was used.Methods: We developed a single-cell model of the physiological germinal center (GC) reaction to describe the dynamics of GC expansion and B-cell affinity maturation in silico. Two different growth compartments (dark and light zone within a GC) are considered. GCs are simulated over time, based on cell-cell and cell-microenvironment interactions. To capture mechanisms such as SHM, we incorporated an artificial genome into each model cell. While IGHV mutations influence antigen affinities, driver mutations are reflected by parameter changes of proliferation and apoptosis behavior in affected cells. Additionally, we permitted cell migration between follicles. Using our model, we simulated primary FL emergence and relapse and compared the results to phylogenetic trees reconstructed from IGHV sequences of paired tumor samples.Results: We identified a set of parameter changes in single cells (driver mutations) which permit an FL-cell population to take over a normal GC, creating a primary tumor in silico. Importantly, our model only provides a consistent explanation for the different types of evolution following tumor initiation if interfollicular cell migration is taken into account. Modulating 2 critical parameters (timepoint of migration and number of migrating cells), the heterogeneity of clonal evolution can be fully reproduced. While "divergent evolution" is only obtained with early migration (i.e., shortly after GC formation) and few migrating FL cells, "sequential evolution" is found in cases with late migration. In contrast, "no evolution" can only be explained if migration of multiple cells is assumed.Our model results are also in agreement with quantitative measurements such as clone sizes and the ratio of replacement and silent IGHV mutations. Additionally, our model is consistent with the observed co-evolution of driver mutations.Finally, we can make biologically and clinically testable model predictions: Upon clonal dominance of FL cells within a GC we predict antigen affinity-independent growth regulation of B-cells and complete cessation of SHM. Furthermore, we predict a pivotal role of cell migration in FL evolution and the existence of extrafollicular niche-like environments in which FL cells lie dormant for extended periods of time. Our simulations indicate that there are no distinct types of evolution. We rather propose to view evolutionary heterogeneity as a continuum, predicting the possibility of co-existence of different patterns of evolution within a patient. This notably also includes the option that the evolutionary sequence of primary and relapse tumor appears reversed.Conclusions: Our work represents the first comprehensive dynamical in silico lymphoma model, providing a consistent explanation for evolutionary heterogeneity. We conclude that the type of evolution is not predictable based on profiling of tumor cells (e.g., with respect to driver mutations) but is rather stochastic (i.e., based on random timepoints and the frequency of cell migration). We therefore caution to draw clinically relevant conclusions from evolutionary profiles. As migration of FL cells within the lymphatic system appears to be a key event in disease evolution, inhibition of cell migration may be a therapeutic target.Acknowledgment: BMBF/PTJ 031 6166 DisclosuresNo relevant conflicts of interest to declare.

Mutations in early follicular lymphoma progenitors are associated with suppressed antigen presentation

Follicular lymphoma (FL) is incurable with conventional therapies and has a clinical course typified by multiple relapses after therapy. These tumors are genetically characterized by B-cell leukemia/lymphoma 2 (BCL2) translocation and mutation of genes involved in chromatin modification. By analyzing purified tumor cells, we identified additional novel recurrently mutated genes and confirmed mutations of one or more chromatin modifier genes within 96% of FL tumors and two or more in 76% of tumors. We defined the hierarchy of somatic mutations arising during tumor evolution by analyzing the phylogenetic relationship of somatic mutations across the coding genomes of 59 sequentially acquired biopsies from 22 patients. Among all somatically mutated genes, CREBBP mutations were most significantly enriched within the earliest inferable progenitor. These mutations were associated with a signature of decreased antigen presentation characterized by reduced transcript and protein abundance of MHC class II on tumor B cells, in line with the role of CREBBP in promoting class II transactivator (CIITA)-dependent transcriptional activation of these genes. CREBBP mutant B cells stimulated less proliferation of T cells in vitro compared with wild-type B cells from the same tumor. Transcriptional signatures of tumor-infiltrating T cells were indicative of reduced proliferation, and this corresponded to decreased frequencies of tumor-infiltrating CD4 helper T cells and CD8 memory cytotoxic T cells. These observations therefore implicate CREBBP mutation as an early event in FL evolution that contributes to immune evasion via decreased antigen presentation.

BCL2 mutation spectrum in B‐cell non‐Hodgkin lymphomas and patterns associated with evolution of follicular lymphoma

BCL2 is a target of somatic hypermutation in t(14;18) positive and also in a small fraction of t(14;18) negative diffuse large B-cell lymphoma (DLBCL), suggesting an aberrant role of somatic hypermutation (ASHM). To elucidate the prevalence of BCL2 mutations in lymphomas other than DLBCL, we Sanger-sequenced the hypermutable region of the BCL2 gene in a panel of 69 mature B-cell lymphomas, including Richter's syndrome DLBCL, marginal-zone lymphomas, post-transplant lymphoproliferative disorders, HIV-associated and common-variable immunodeficiency-associated DLBCL, all known to harbour ASHM-dependent mutations in other genes, as well as 16 t(14,18) negative and 21 t(14;18) positive follicular lymphomas (FLs). We also investigated the pattern of BCL2 mutations in longitudinal samples from 10 FL patients relapsing to FL or transforming to DLBCL (tFL). By direct sequencing, we found clonally represented BCL2 mutations in 2/16 (13%) of t(14;18) negative FLs, 2/16 (13%) HIV-DLBCLs, 1/9 (11%) of Richter's syndrome DLBCL, 1/17 (6%) of post-transplant lymphoproliferative disorders and 1/2 (50%) common-variable immunodeficiency-associated DLBCL. The proportion of mutated cases was significantly lower than in FLs carrying the t(14;18) translocation (15/21, 71%). However, the absence of t(14;18) by FISH or PCR and the molecular features of the mutations strongly suggest that BCL2 represents an additional target of ASHM in these entities. Analysis of the BCL2 mutation pattern in clonally related FL/FL and FL/tFL samples revealed two distinct scenarios of genomic evolution: (i) direct evolution from the antecedent FL clone, with few novel clonal mutations acquired by the tFL major clone, and (ii) evolution from a common mutated long-lived progenitor cell, which subsequently acquired distinct mutations in the FL and in the relapsed or transformed counterpart.

A Comparative Analysis Of Next-Generation Sequencing and Real-Time Quantitative PCR For Minimal Residual Disease Detection In Follicular Lymphomas

BackgroundDetection of minimal residual disease (MRD) by t(14;18) Real-Time Quantitative (RQ) PCR is an important tool during clinical follow-up in patients (pts) with follicular lymphoma (FL). However, only the major breakpoint region (MBR) and minor cluster region (mcr) breakpoints have been currently exploited as MRD targets. Therefore, only 50% to 65% of pts can be assessed by t(14;18) RQ-PCR. Alternative targets such as the immunoglobulin heavy chain variable region (IGH) can be used only with limitations due to somatic hypermutation. IGH-based next-generation sequencing (NGS) might provide an alternative approach and provide increased sensitivity, specificity, accuracy and reproducibility. We performed a comparison of the two approaches in 29 FL pts. Patients and MethodsOverall, 206 samples (85 bone marrow, 114 peripheral blood, 6 stem cell aliquots and one lymph node sample) were investigated from 29 FL pts in which RQ-PCR based MRD analysis had been performed in the context of prospective clinical trials. Overall, 33 dx and 173 follow-up (FU) samples were analyzed. 23/29 pts had a PCR detectable t(14;18) rearrangement, 5 pts had a clonal IGH rearrangement only and one patient had no marker by consensus PCR. NGS was performed at Sequenta in South San Francisco. Using universal primer sets, we amplified IGH variable, diversity, and joining gene segments from genomic DNA. Amplified products were sequenced to obtain a high degree of coverage and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency in the dx sample and then quantitated in FU samples.Overall comparability of MRD results by RQ-PCR and NGS was assessed using correlation analysis. A positive/negative discordance between two results was defined as major when the positive result was >1E-05 and minor when ≤1E-05; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log. ResultsAll 29 pts were evaluable with at least one method. IGH NGS identified an index clone in 22/29 cases. Seven patients pts were only quantifiable by t(14;18) RQ-PCR while IGH NGS did not identify an index clone for sequencing. In all but one dx sample demonstrated low level lymphoma infiltration with MRD level below 10-3. In one of these cases, IG kappa could be successfully sequenced for MRD indicating that not only low level MRD but also somatic mutation of IGH is a potential pitfall for MRD detection by NGS in FL.15 pts were evaluable for MRD by t(14;18) RQ-PCR and NGS. In 97 FU samples, a significant concordance between MRD methods could be demonstrated (r2=0.80) (p<0.0001). Of these samples, 44 were MRD positive and 45 were MRD negative with both tools. A quantitative discordance occurred in 12/44 MRD positive samples, where MRD was higher in 7 samples and lower in 5 samples by NGS. A major discordance occurred in 4 samples where t(14;18) RQ-PCR was positive and NGS was negative. A minor discordance was detectable in 4 samples where in 2 samples t(14;18) RQ-PCR was positive and NGS was negative, and in the other 2 samples the opposite was correct.In 5 t(14;18) negative cases and one with an unusual large t(14;18) rearrangement MRD was quantified by IGH-RQ-PCR using cloned plasmids and IGH NGS. In all cases both methods also showed excellent concordance.One advantage of the IGH NGS approach is its ability to detect different IGH sequences generated by the somatic hypermutation process. We have seen in some patients a dynamic picture of the IGH clonotypes with the rise and fall of different clonotypes at different time points (Fig 1). [Display omitted] ConclusionsNGS represents a feasible tool for IGH-based MRD monitoring that allows analysis of a larger group of FL pts. Our results show that the two methods have a high level of correlation. Lymphoma infiltration of dx samples and somatic mutation of IGH is a critical point for identification of the tumor-specific clonotypes by NGS, therefore different MRD methods should complement each other to allow MRD assessment for the majority of pts. Furthermore IGH NGS sequencing has the potential to detect and track IGH evolution in FL. Disclosures:Kneba:Roche: Consultancy, Research Funding. Faham:Sequenta, Inc.: Employment, Patents & Royalties.

Early lesions of follicular lymphoma: a genetic perspective

The pathogenesis of follicular lymphoma is a multi-hit process progressing over many years through the accumulation of numerous genetic alterations. Besides the hallmark t(14;18), it is still unclear which other oncogenic hits contribute to the early steps of transformation and in which precursor stages these occur. To address this issue, we performed high-resolution comparative genomic hybridization microarrays on laser-capture micro-dissected cases of follicular lymphoma in situ (n=4), partial involvement by follicular lymphoma (n=4), and duodenal follicular lymphoma (n=4), assumed to represent, potentially, the earliest stages in the evolution of follicular lymphoma. Cases of reactive follicular hyperplasia (n=2), uninvolved areas from follicular lymphoma in situ lymph nodes, follicular lymphoma grade 1-2 (n=5) and follicular lymphoma grade 3A (n=5) were used as controls. Surprisingly, alterations involving several relevant (onco)genes were found in all entities, but at significantly lower proportions than in overt follicular lymphoma. While the number of alterations clearly assigns all these entities as precursors, the pattern of partial involvement by follicular lymphoma alterations was quantitatively and qualitatively closer to that of follicular lymphoma, indicating significant selective pressure in line with its faster rate of progression. Among the most notable alterations, we observed and validated deletions of 1p36 and gains of the 7p and 12q chromosomes and related oncogenes, which include some of the most recurrent oncogenic alterations in overt follicular lymphoma (TNFRSF14, EZH2, MLL2). By further delineating distinctive and hierarchical molecular and genetic features of early follicular lymphoma entities, our analysis underlines the importance of applying appropriate criteria for the differential diagnosis. It also provides a first set of candidates likely to be involved in the cascade of hits that pave the path of the various progression phases to follicular lymphoma development.

Genomic analysis reveals epigenetic ‘addiction’ underpinning follicular lymphoma and its transformation – a rationale for targeted epigenetic therapies

Follicular lymphoma (FL) continues to pose a clinical challenge with a progressive disease course typified by multiple relapses, eventual resistance to standard therapies and transformation (tFL) in a subset of patients to the more aggressive diffuse large B cell lymphoma (DLBCL). WGS was performed on 6 paired FL-tFL patients and corresponding germ-lines to understand the clonal dynamics of progression and recurring genetic events in FL. We identified that the dominant pattern of FL evolution to transformation was consistent with the existence of a common origin, an ancestral common progenitor cell (CPC) population. Strikingly, the CPCs were enriched for mutations in chromatin regulatory genes with every case harbouring mutations in MLL2, a histone methyltransferase, concurrent with mutations in other methyltransferases (EZH2, MLL3, PRMT2), acetyltransferases (CREBBP, MEF2B), bromodomain proteins (BRD2, CECR2), core and linker histones. Targeted deep sequencing of EZH2 showed mutations in 27% of cases in an extension cohort of 366 cases, much higher than previously reported. All mutations targeted the catalytic SET domain leading to a gain-of-function thus offering the possibility of using EZH2 inhibitors. The plethora of genetic mutations in epigenetic regulators in FL shown in this study therefore offers a compelling model disease to strategically test epigenetically-targeted therapies.

Quantitative Ultra-Deep Sequencing of Non-Coding Regions to Characterize Clonal Evolution in Follicular Lymphoma

Abstract 146 Background:Cancers are characterized by genomic instability, and the resulting intra-clonal diversity is thought to underlie the evolution of more aggressive clinical course behavior and therapy resistance. Intra-clonal heterogeneity in follicular lymphoma (FL) has been documented qualitatively by analysis of mutations induced by Activation Induced Deaminase (AID) in IGH coding regions. AID also aberrantly targets non-coding regions of the genome causing numerous “passenger” mutations. To obtain a quantitative measure of genetic variation and to characterize population dynamics in FL, we performed ultra-deep “next generation” sequencing of selected non-coding regions known to be aberrantly targeted by AID in FL. Herein, we quantify and characterize AID-induced genetic variation in FL and relate the extent of variation to grade. Method:An ultra-deep sequencing approach, able to identify sequences present at 0.3% frequency, was developed to quantify sub-clonal populations within a tumor so that the extent of intraclonal heterogeneity can be compared between tumors. Genomic DNA was obtained from fresh/frozen specimens (10 FL grade 1/2, 2 FL grade 3, 3 hyperplastic lymph nodes and 1 epithelial cell line). H&E sections and flow cytometry data were examined to corroborate the diagnosis and fraction of tumor (>50% in all cases). The ten sequenced (ABI-SoLID) regions were PCR amplicons of: 5’UTRs of BCL2, BCL6, MYC, PAX5, PIM1, RHOH, CD83 and SYK; IGH (mu enhancer-J6; clone-specific Vh-J6). Average mapped coverage was > 20,000-fold. To detect rare mutations while suppressing read noise, data were mapped using an algorithm (BFAST) designed to detect rare Single Nucleotide Variants (SNV); a novel recursive mapping pipeline enhanced detection of SNVs in heavily mutated regions. A novel filtering algorithm handled data as 34 base-pair “words” with confirmation based on partial assembly using independently obtained sequences from adjacent regions. Results:656 mutations were detected in non-coding regions of the 12 FL specimens. FL specimens had more mutations in all 5’ UTRs combined (range 14–193, median 47, n=12) than control specimens (range 2–7, n=4). The mutations were associated with AID-motifs (p<0.05). 8 regions (excluding SYK and MYC) had significantly more SNVs in FLs compared to controls. BCL2 had the largest number of SNVs and the number of SNVs in BCL2 predicted the sum of SNVs in the 9 other regions (r2=0.85). The specimens could be placed into 4 strata based on the number of BCL2 SNVs (Poisson ladder, p=0.005). No correlation was seen between the number of SNVs and grade, percent nodularity, percent tumor, or other histologic features. Of the 354 SNVs in BCL2, many were “rare”: 78 were present in less than 1% of alleles and 41 in less than 0.5%. Strikingly, all low frequency CNVs were found in cases of Grade 1/2 FL without any found in either of the Grade 3 FL specimens despite the abundance of higher frequency CNVs in these cases. Representing the data as evolutionary trees suggested that the grades have differing population dynamics: Grade 1/2 showed dominant populations from which similarly proliferative subpopulations were continuously derived while Grade 3 showed a dominant population with remnant evolutionarily antecedent cells but little evidence of continuing development of intraclonal variation. Conclusions:The number of SNVs in AID-targeted non-coding regions varied widely allowing stratification of FL cases into 4 groups. The number of SNVs in BCL2 non-coding regions correlated with the number of SNVs in the other 8 gene regions combined, suggesting that analysis of the BCL2 non-coding region is sufficient to characterize the genome-wide effects of aberrantly targeted AID. Furthermore, low frequency mutations (present in less than 10% of sequences) were found only in grade 1/2 but not in grade 3. Representing the data as evolutionary trees indicated that population dynamics also varied greatly, with evidence that in low grade FL there is active ongoing generation of diversity while in grade 3 FL the population is dominated by the most evolutionarily derived clone with relatively less ongoing diversification. In contrast to prior methods, this quantitative approach to tumor evolution may allow correlation of intraclonal genetic diversity with natural history, response to immune-chemotherapy and risk of transformation to diffuse large cell lymphoma. Disclosures:No relevant conflicts of interest to declare.

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

AbstractAbstract 680Pathogenesis 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.

Clinical spectrum and prognosis of follicular lymphoma with blastoid transformation: case series and a review of the literature

Follicular lymphoma (FL) with blastoid transformation (FL-BT) is a rare entity. FL-BT has a dismal prognosis due to intrinsic biological characteristics, lack of evidence-based therapeutic guidelines, and exclusion from clinical trials. In order to understand the natural course of the disease and reconcile treatment strategies in patients with FL-BT, we reviewed available data. Thirty patients were identified, three from our institution (Roswell Park Cancer Institute) and 27 patients from peer-reviewed literature. Demographic, clinical, treatment, pathological, and cytogenetic characteristics were described. Most of the patients (90%) were treated with chemotherapy at transformation. Complete and partial responses to treatment were observed in 38.9% and 22.3% of the patients, respectively. The median overall survival for the entire group of patients was 11 months (range 0.8-30 months); half of the patients were found to have concurrent genetic alterations in the BCL-2 and C-MYC gene rearrangements (double hit). FL-BT with a double hit had a shorter median overall survival when compared to nondouble hit FL-BT (7 months vs. 26 months, respectively, P = 0.005). In our experience, FL-BT is a rare and grave clonal evolution in FL characterized by an aggressive clinical course, inferior response to standard chemoimmunotherapy, and poor clinical outcome. C-MYC rearrangements are common in FL-BT and may play a role in the progression from FL to FL-BT or the poor clinical outcomes observed. Periodic updates from academic institutions treating FL-BT may improve patient care.