Biology Of Follicular Lymphoma Research Articles

Recent advances in understanding the biology of follicular lymphoma.

Follicular lymphoma (FL), the most common indolent B-cell lymphoma, develops over decades before manifesting as overt disease. BCL2 overexpression by t(14;18) confers a survival advantage to B cells during the germinal center reaction, and abnormalities in epigenetic modifier genes lead to desynchronization of gene expression changes in germinal center B cells. Studies in mouse models have shown that BCL2 overexpression and epigenetic deregulation in B cells cooperatively promote lymphomagenesis. The immune microenvironment also plays an essential role in the biology of FL, and many molecular prognostic indicators based on the immune microenvironment have been proposed. However, high-risk gene signatures do not appear to be consistent between patients receiving different chemotherapies. FL cells frequently carry N-linked glycosylation motifs within the immunoglobulin gene, leading to chronic activation of the B-cell receptor (BCR). Recent evidence suggests that this chronic BCR signaling drives FL polarization toward a dark-zone phenotype and promotes clonal evolution. Since both epigenetic and post-transcriptional modifications of B cells have been implicated in the early stage of FL development, it may be possible to use novel non-chemotherapeutic approaches that interfere with the immunobiology in treatment or early prevention of FL.

Preliminary Analysis of an Ongoing Phase II Study of Response-Adapted Therapy with Copanlisib and Rituximab for Untreated Follicular Lymphoma

Introduction Frontline therapy for pts with follicular lymphoma (FL) is not based on the molecular profile of the tumor. The PI3K pathway is central to FL biology, but the molecular profile of tumors most sensitive to PI3K inhibition (PI3Ki) is unknown. Copanlisib inhibits both PI3Kα and δ isoforms, and we hypothesized that most pts with treatment naïve FL will have PI3Ki-sensitive tumors and durable complete responses (CRs) may be achievable with short durations of targeted therapy. A gene expression profiling (GEP) predictor utilizes a combination of the expression of 45 selected informative genes and an additional 19 housekeeping genes to identify FL pts at high-risk for early progression within 24 months (Wright et al. ASH Annual Meeting2022). We also hypothesized that copanlisib would induce durable CRs in high-risk pts by the GEP assay. Here, we report a preliminary analysis of an ongoing “window of opportunity” study of copanlisib followed by response-adapted copanlisib and rituximab in treatment-naïve pts with FL (NCT03789240). Methods Pts with untreated grade 1-2, 3A FL, ≥stage 2 are eligible if systemic therapy is indicated: symptoms, progressive lymphadenopathy, or organ compromise. Prior radiation is permitted. Eligibility includes age ≥18 and adequate organ function unless due to lymphoma. HIV, CMV, Hep B or C, and autoimmune conditions are excluded. Pts first receive copanlisib 60mg on days 1, 8, and 15 of a 28-day “window of opportunity” to explore the activity of copanlisib. Following the window, pts receive 6 cycles of copanlisib 60mg on days 1, 8, and 15 of a 28-day cycle along with rituximab 375mg weekly x 4 then on day 1 of each cycle and response is assessed with FDG-PET and CT scans. Pts without response after C6 are taken off study and those with a partial response (PR) can continue for an additional 6 cycles and then stop therapy. Pts who achieve a CR after 6 cycles stop therapy. No maintenance is given. Supportive care includesPCP prophylaxis. The primary endpoint is the CR rate with secondary endpoints of safety, duration of CR, and PFS. Exploratory objectives include identification of a predictive signature of PI3Ki response and the response rate in high-risk pts identified by the GEP assay. Results Twenty-eight pts have enrolled. Median age was 56y (range, 24-80). Seventeen (61%) and 12 (43%) pts had high-risk FLIPI and FLIPI-2 scores ≥3, respectively; 6 (21%) pts had Grade 3A FL, and 17 (61%) pts met two or more GELF criteria. After copanlisib monotherapy in the window, 27 (96%) pts had tumor reductions by CT with a median change of -38% (-62% to +16%) (Figure 1). Among 22 pts who completed 6 cycles, 21 (96%) responded, including 9 (41%) pts with PR and 12 (55%) pts with CR. Of 9 pts with PR who received an additional 6 cycles of therapy, only 1 (11%) converted to a CR bringing the overall CR rate to 59% (13/22 pts). After a median follow-up of 25.8 months, the 2-year PFS was 43.2% (95% CI, 20.7-64.0%). The median duration of CR was 29.7 months (95% CI, 6.3 months - not estimable) with 8 (62%) CRs ongoing (Figure 2). Toxicity was evaluated in 28 pts across 220 cycles. Most common were rash (64%), mucositis (50%), and nausea/vomiting (50%), all G1-2 except 1 (4%) pt with G3 mucositis. The most common ≥G3 toxicities were neutropenia and infections in 5 (18%) pts each. ≥G3 neutropenia occurred in 10 (5%) cycles, responded to therapy interruption and/or growth factors, and did not require dose reduction. Two (8%) pts had copanlisib dose reduced, 1 (4%) each due to rash and mucositis. Therapy was prematurely discontinued in 5 pts: 3 due to COVID-19, 1 for hepatotoxicity that resolved, and 1 due to infusion reaction to rituximab. Nine of 24 (38%) pts were poor risk by the GEP assay. Complete responses were observed in 71%, 67%, and 57% of pts categorized as high-risk by FLIPI, FLIPI-2, and GEP assay, respectively. The CR rate was not affected by bulky disease (any LN >7cm; p=1.00), high-risk FLIPI (p=0.19), high-risk FLIPI-2 (p=0.67), poor-risk GEP (p=1.00), or metabolic tumor volume (p=0.33). Conclusion Nearly all pts with treatment naïve FL have PI3Ki-responsive tumors, and the combination of copanlisib with rituximab achieved a CR rate of 59% at the end of induction therapy. The median duration of CR is nearly 30 months, including ongoing remissions without maintenance. CRs were observed in pts deemed high-risk by the GEP assay and FLIPI score. The safety profile is excellent, with mostly G1-2 toxicities manageable with supportive care.

Follicular lymphoma microenvironment: insights provided by single-cell analysis.

Follicular lymphoma (FL) is the most frequent indolent lymphoma and is characterized by the abundant infiltration of tumor microenvironment (TME) cells. The activity of TME cells reportedly plays an important role in the biology of FL. TME cells that reside within neoplastic follicles, such as T-follicular helper cells and follicular dendritic cells, have been shown to aid in FL development and progression through interactions with malignant B cells, whereas regulatory T cells have unexpectedly shown an apparently favorable prognostic impact in FL. Unfortunately, the understanding of the FL TME, particularly regarding minor cell subsets, has been hampered by unknown cell heterogeneity. As with other solid and hematologic cancers, novel single-cell analysis technologies have recently been applied to FL research and have uncovered previously unrecognized heterogeneities, not only in malignant B cells but also in TME cells. These reports have greatly increased the resolution of our understanding of the FL TME and, at the same time, raised questions about newly identified TME cells. This review provides an overview of the unique aspects of FL TME cells with a clinical viewpoint and highlights recent discoveries from single-cell analysis, while also suggesting potential future directions.

Biology of follicular lymphoma: insights and windows of clinical opportunity.

Follicular lymphoma (FL) is a heterogeneous disease, both clinically and biologically. The biological behavior and development of FL is a culmination of complex multistep processes underpinned by genetic and nongenetic determinants. Epigenetic deregulation through recurrent genetic alterations is now a recognized major biological hallmark of FL, alongside the t(14;18) translocation. In parallel, there is a strong interplay between the lymphoma B cells and the immune microenvironment, with the microenvironment serving as a critical enabler by creating a tumor-supportive niche and modulating the immune response to favor survival of the malignant B cells. A further layer of complexity arises from the biological heterogeneity that occurs between patients and within an individual, both over the course of the disease and at different sites of disease involvement. Altogether, taking the first steps to bridge the understanding of these various biological components and how to evaluate these clinically may aid and inform future strategies, including logical therapeutic interventions, risk stratification, therapy selection, and disease monitoring.

Architecture and Early Construction of a Multi-Modal Oncology Registry for Follicular Lymphoma

Background: Follicular Lymphoma (FL) is the most common indolent lymphoma. While firm data are lacking, global prevalence estimates are >1 million. FL is clinically heterogeneous. Though initial therapy varies, FL responds in most patients, but is not cured. With multiple relapses after variable treatments, each individual patient journey differs. For the 20% of cases relapsing within two yrs (POD24), prognosis is unfavorable, with 5-yr survival ~50%. A stochastic risk of 2-3%/yr for transformation to aggressive lymphoma carries anxiety and poor outcomes when it occurs. Reproducible predictive biomarkers for POD24 or transformation are lacking. FL is also biologically heterogeneous, with varied epigenetic changes cooperating with the t(14;18) translocation dysregulating BCL2. Given clinical and biological heterogeneity and added complexity of variable treatments, the unmet need of better understanding the underlying biology of FL would benefit from a large comprehensive and integrated clinical and biological database. To garner such new information in FL warrants a new approach to data collection, aggregation, and analysis, needed to generate Real World Evidence (RWE) with longitudinal data collection. Methods: The Follicular Lymphoma Foundation (FLF) undertook an assessment based on input from multiple stakeholders (patients, clinicians, researchers, life sciences companies), culminating in a workshop to identify key unmet needs in FL and define ways philanthropy could best address these. Key areas: optimizing treatment regimens and sequence; cumulative toxicity profiling and prediction; biomarkers for functional cure; and treatment-centric RWE. The output suggested creating a Precision Medicine Program. This direct-to-patient clinical and biodata registry will be the largest multi-modal registry for patients with FL. Early-stage work in architecting the database models, what data can and will be collected and how it should be stewarded is in progress. We are leveraging experiences of the MMRF (CureCloud®) to accelerate our progress. In addition to clinical and biodata, a biobank for plasma and remnant tissue will be developed. Collecting and aggregating data from varied data sources (Electronic Medical Records (EMR) abstraction, molecular profiling to include genomic, proteomic, and epigenetic data, immune profiling, and patient-reported outcomes) will provide a longitudinal 360-degree view of FL, integrated with outcomes. We continue to seek expert stakeholder input to ensure this comprehensive database will be as broadly useful as possible. Trial Planning: An IRB-approved clinical study is being designed to permit patients (initially U.S. with later international expansion) to enroll on the FLF website via electronic consent which will allow EMR extraction and collection of any existing tissue. Patients will be asked to supply Patient Reported Outcome (PRO) data serially on validated questionnaires. Existing tissue samples will be sought from all patients for analysis to establish a cumulative molecular database; tissue is not required for enrollment. Specific scientific aims are to use clinical and biologic data to: Optimise treatment care pathways; Profile and predict treatment toxicity; Develop surrogate endpoints for durable response; Enable innovative trial designs. Eligible patients will be at least 18 yrs with diagnosis of FL regardless of stage or prior treatment. A Patient Support Center will assist registrants during and subsequent to enrollment and to maintain engagement. Statistical analysis will mainly be descriptive, based on expected accrual, incorporating multiple imputation techniques for missing data. Data security will be incorporated. Initial pilot of 100 cases from an existing tissue bank with associated EMR data will precede the main direct-to-patient effort. Discussion: The registry expects > 5000 accruals in the first 6 years. The initial data set of 500-1000 patient cases of FL with molecular and EMR matched data to begin the journey to identify novel biomarkers and molecular targets is expected to be available in 3-5 years. The FLF will report registry construction progress as the program advances. This program fills a unique position of prospectively acquired RWE including biological data as a resource to help identify new biomarkers, drug targets, and facilitate a new era of personalized medicine in FL treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prediction of Early Disease Progression at 24 Months (POD24) Using Pre-Treatment Biopsies from Patients with Follicular Lymphoma (FL) Treated with Immunochemotherapy (IC)

Background: Gene expression profiling has been successfully used to interrogate FL biology related to overall survival alone or in combination with clinical parameters and mutation profiles such as the M7-FLIPI. However, none of these has ever been specifically trained against the most reproducible and pertinent predictor of excess mortality: early progression of disease by 24 months (POD24). Nor has any assay been validated on a clinical platform for testing patients in a CAP-CLIA environment. We therefore undertook a discovery analysis and also investigated published gene signatures to determine the best method for upfront prediction of POD24 with clinical progression (not transformation). Methods: For model generation, we assembled and harmonized gene expression data from frozen tissues from 377 total FL cases treated with immunochemotherapy (IC) from the Lymphoma/Leukemia Molecular Profiling Project (LLMPP; N=80, unpublished, Affymetrix), British Columbia (N=137, Silva et al, 2018, DASL Illumina), and PRIMA cohorts (N=160, S. Huet et al, 2018, Affymetrix) and performed in silico analysis to identify the most promising candidate genes to predict POD24. In addition to the identified genes, we also included all "IR1/IR2" genes (S. Dave 2004) and "PRIMA23" (Huet) genes and other published genes of interest totaling 144 genes related to tumor, microenvironment, and housekeeping functions. We used these 144 genes as the basis for an nCounter code set (NanoString, Seattle, WA), and then evaluated their expression in formalin-fixed, paraffin-embedded (FFPE) tissues from a training set of 57 matching cases from the LLMPP cohort and an independent set of 59 independent samples from the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). Comparing the FFPE NanoString results from the LLMPP training set to their matched fresh frozen Affymetrix results identified, 48 of the 144 candidate genes for which the two platforms showed agreement with correlation > 0.5). Multiple prediction architectures were compared using these 48 genes and optimized via cross-validation on this training set to develop a final locked down POD24 predictor ("CCP-32") based on a linear combination of the expression of 32 selected informative genes and an additional 19 housekeeping genes. Additionally, for comparison, the Huet profiling scores were calculated on the LLMPP training samples and a cut-point, optimized to select patients with POD24 as a dichotomous endpoint. These two predictors were then applied to an independent set of 174 FFPE samples from the MER population-based cohort. Samples with very low signal in the normalization genes were excluded (8/116 training and 17/174 validation). Samples from patients that were event free until they experienced transformation or were lost to follow-up in less than 24 months after IC were treated as censored and were thus excluded (2/116 training, 6/174 validation). Results: When applied to the MER FFPE validation set, the CCP-32 predictor placed 30% in a poor prognosis group with an observed POD24 rate of 56% while the remaining 70% samples had an observed 18% POD24 - an enrichment that was statistically significant (p=2.9x10-5). A training-optimized cut of the Huet profiling score performed similarly on the validation set with 44% placed in the poor prognosis group, with an observed POD24 rate of 48%, while the remaining 56% had an observed POD24 of 15% (p=1.5x10-5) Conclusions: Starting with an agnostic in silico analysis of data from frozen FL tissue to identify predictive genes, also including known gene sets pertinent to FL, we first identified genes that correlated best in FFPE tissues using digital expression profiling to develop an algorithm specifically trained to detect POD24 due to progression (not transformation) on a clinical platform using routinely processed samples. A novel predictor and the Huet profiling score were each able to detect a biological subset that was heavily enriched in the approximately 30-44% of FL patients at risk for POD24. Additional cohort analyses are underway. Integration of gene mutations and other genetic factors will also be explored to determine whether these can improve the predictive value with a long-term goal of creating a combined assay that could be suitable for use in a clinical laboratory to select high risk patients for clinical trials.

Follicular Lymphoma: a Focus on Current and Emerging Therapies

Follicular lymphoma (FL) is the most common indolent lymphoma and is characterized by a relapsing and remitting course. In addition to significant biologic heterogeneity, the clinical trajectory for patients is variable, with some being observed for many years, and others having aggressive disease requiring multiple treatment courses. Unfortunately, FL remains incurable, and continues to cause early mortality. Improved understanding of the genetic and immune biology of FL has led to several FDA-approved therapies in the relapsed and refractory setting, including PI3K inhibitors; immunomodulatory agents; the EZH2 inhibitor, tazemetostat; and anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, axicabtagene ciloleucel. This review outlines the current approach to the diagnosis and treatment of FL with a focus on emerging investigational therapies, including targeted protein inhibitors, antibody-drug conjugates, monoclonal antibodies, bispecific antibodies, and novel combination strategies.

Targeted Therapy for Relapsed/Refractory Follicular Lymphoma: Focus on Clinical Utility of Tazemetostat.

The management of follicular lymphoma (FL) in the relapsed and refractory setting is challenging and an area of ongoing investigation. Epigenetic dysregulation has recently been shown to be a hallmark of FL. Mutations in histone-modifying genes are likely early, driver events in FL pathogenesis, and so are attractive targets to drug. Gain-of-function mutations in the histone methyltransferase EZH2 are common in FL and maintained through disease evolution. With mounting data supporting a critical role for EZH2 as an oncogenic driver for FL, the small molecule inhibitor, tazemetostat, was developed. Tazemetostat has shown promising activity in preclinical models and early phase trials. Importantly, responses were seen in patients with high-risk features. Based on these data, tazemetostat was approved in the US in 2020 for EZH2mut patients with FL who had received at least two prior lines of systemic therapy, or for EZH2wt patients without alternative treatment options. Here, we will review the biology of FL as it pertains to tazemetostat, the available clinical trial data, and future directions for this new therapy.

Follicular Lymphoma: a Focus on Current and Emerging Therapies

Follicular lymphoma (FL) is the most common indolent lymphoma and is characterized by a relapsing and remitting course. In addition to significant biologic heterogeneity, the clinical trajectory for patients is variable, with some being observed for many years, and others having aggressive disease requiring multiple treatment courses. Unfortunately, FL remains incurable, and continues to cause early mortality. Improved understanding of the genetic and immune biology of FL has led to several FDA-approved therapies in the relapsed and refractory setting, including PI3K inhibitors; immunomodulatory agents; the EZH2 inhibitor, tazemetostat; and anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, axicabtagene ciloleucel. This review outlines the current approach to the diagnosis and treatment of FL with a focus on emerging investigational therapies, including targeted protein inhibitors, antibody-drug conjugates, monoclonal antibodies, bispecific antibodies, and novel combination strategies.

Follicular lymphoma: the diminishing role of chemotherapy

Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma, accounting for 70% of cases in Western countries. Despite this unique name, FL is an extremely heterogeneous disease, both clinically and biologically. The basis of FL heterogeneity lies in the different biological pathways which can be activated, because of the variety of gene mutations that can occur. Today, there is a growing interest in the knowledge of these activated pathways, which is also testified by the presence of a new model that incorporates FL mutations to define patient’s prognosis (m7-FLIPI). These evaluations are also appealing because of the recent possibility of using “targeted therapies”. Targeted therapies are new tools, currently applicable in the setting of relapse/refractory (R/R) disease, where we can find a great variety of “chemo-free” combinations. As in other hematologic malignancies, “cellular therapy” enriches FL drug scenario, including T-cell dependent bispecific antibodies and chimeric antigen receptor (CAR) T-cell. Since FL heterogeneity is the basis of the difference in therapeutic efficacy and disease course among patients, the hope for the future is to understand FL biology more deeply, to better comprehend how to obtain more representative samples and pre-treatment prognostic information in order to individualize the treatment strategy as early as frontline therapy

Cytomegalovirus (CMV) Infection Suppresses The Expression Levels of Serum Cytokines in Follicular Lymphoma Patients

Follicular lymphoma (FL) is an indolent B-cell neoplasm. Increasing evidence suggests that chronic cytomegalovirus infection impacts the immunity of FL patients. This study was designed to unveil the impact of CMV infection on FL biology. Pretreatment serum samples from 42 FL patients recruited into a phase 3 FL clinical trial to compare two alternative frontline chemoimmunotherapy regimens were used. Twenty-one (21) of these patients had tested positive for CMV IgG antibodies, whereas 21 patients were negative. A multiplex assay was employed to measure the expression levels of 27 cytokines in each of these samples. Results were then compared between CMV-positive and CMV- negative groups using the Mann-Whitney U test using IBM SPSS Statistics version 20. Sera from CMV-positive FL patients showed decreased expression levels of most cytokines than those from the CMV-negative population. Data shows a statistically significant down-regulation of IL-6, IL-8, IL-9, IL-17A, FGF-basic, MIP-1α, and MIP-1β expression in CMV-positive FL patients compared to the CMV-negative group. Overall, our results suggest that CMV infection down-regulates the expression levels of serum cytokines in FL. Complementary studies to investigate immune cells in these patients' blood and tissue biopsies could provide a comprehensive perspective on the impact of CMV infection on FL.

Follicular Lymphoma Tumor-Cell Transcriptional Programs Associate with Distinct Somatic Alterations and Tumor-Immune Microenvironments

Background: Follicular lymphoma (FL) exhibits significant clinical, cellular, molecular, and genetic heterogeneity. Our understanding of FL biology and molecular classifications of FL, to date, has largely been driven by pathologic classification (Grade 1-3b), genetic classification (m7-FLIPI), or gene expression profiling (IR-1/2; Huet-23), along with limited studies on small cohorts or targeted panels. In order to further understand the biological underpinnings and complexity of FL, large-scale and integrated whole exome sequencing (WES) and RNA sequencing (RNAseq) studies are needed. Using a highly-annotated cohort of 93 FL tumors with matched RNAseq, WES, and CyTOF data, we have explored the transcriptomic signature of purified FL B cells and identified unique molecular subsets that are defined by distinct pathway activation, immune content, and genomic signatures.Methods: Frozen cell suspensions from 93 untreated FL (Grade 1-3b) patients' tumor biopsies, enrolled in the University of Iowa/Mayo Clinic Lymphoma SPORE, were used for the study. DNA was isolated from whole tumor cell suspensions, and RNA was isolated from both whole tumor and B cell-enriched cell suspensions. RNAseq and WES were performed in the Mayo Clinic Genome Analysis Core. RNAseq and WES data were processed using a standard pipeline and novel driver genes were identified using Chasm+ driver analysis. Copy number variants were identified from WES data using GISTIC 2.0. NMF clustering and single sample gene set testing for B cell lineage and tumor microenvironment (TME) signatures were performed in R using the NMF and singscore packages.Results: Unsupervised clustering of RNAseq data identified three distinct expression programs which separated patient B cell samples into 3 groups: Group 1 (G1, n=20), Group 2 (G2, n=24), Group 3 (G3, n=43). While no clinical attributes were defined by any single group, G1 consisted of cases that had less aggressive characteristics (63% Stage I-II, 79% FLIPI 0-1). To identify unique transcriptional pathways driving the three expression programs, we scored gene level contributions to NMF expression programs and employed gene set enrichment analysis. This revealed significant pathway association with type-I IFN signaling (G1), DNA repair and stress response (G2), and epigenetic modulation (G3) as differentiating programs between the 3 groups (FDR q<0.001). VIPER master regulator activity inferencing revealed that these pathways were likely being controlled by differential activity in NF-kB, IRFs, STAT1, BCL6, and FOXO1. Each program significantly enriched for, but were not defined by, portions of specific germinal center programs, such as pre-memory (G1), light-zone-to-dark-zone transition (G2), and a pre-light-zone intermediate (G3). We next assessed the connection between B cell programs and the tumor microenvironment (TME) using available paired CyTOF data on 67 cases, which revealed an active TME in G1, with an abundance of CD8 T cell and NK cell populations, a wide variety of immune content in G2 that consisted mostly of Tfh and myeloid cells, and a poorly populated immune compartment in G3 compared to G1 and G2. Finally, somatic driver mutations and copy number alterations from WES were identified and associated with the three clusters. The three groups distinguished themselves by significant enrichment of copy number alterations (TNFAIP3-loss , 1q23-gain, 1q32-gain) in G2, while 10q-loss and mutations in BCL2 and chromatin modifiers (KMT2D and CREBBP) enriched in G3. G1, overall, had lower alteration burden and had weak associations with any specific alterations, suggesting an alternative mechanism for driving the G1 program.Conclusion: In this study, we have identified three unique FL tumor B cell groups, defined by specific transcriptional programs. Pathways such as inflammation, DNA damage response, and chromatin modification contribute most to the differences between B cell samples and group membership. Additionally, each program associated with specific genetic events as well as TME composition, highlighting potential drivers of these B cell states. This study improves the understanding of the mechanisms driving FL tumors and motivates further investigation into transcriptional consequences of genetic events as well as potential tumor intrinsic factors that may influence the TME. [Display omitted] DisclosuresMaurer: BMS: Research Funding; Genentech: Research Funding; Morphosys: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Nanostring: Research Funding. Rimsza: NanoString Technologies: Other: Fee-for-service contract. Link: MEI: Consultancy; Genentech/Roche: Consultancy, Research Funding; Novartis, Jannsen: Research Funding. Habermann: Tess Therapeutics: Other: Data Monitoring Committee; Seagen: Other: Data Monitoring Committee; Incyte: Other: Scientific Advisory Board; Morphosys: Other: Scientific Advisory Board; Loxo Oncology: Other: Scientific Advisory Board; Eli Lilly & Co.,: Other: Scientific Advisor. Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. King: Celgene/BMS: Research Funding. Cerhan: Genentech: Research Funding; Regeneron Genetics Center: Other: Research Collaboration; Celgene/BMS: Other: Connect Lymphoma Scientific Steering Committee, Research Funding; NanoString: Research Funding. Novak: Celgene/BMS: Research Funding.

Characterization of the Immunogenomic Landscape of Follicular Lymphoma

Background: Follicular Lymphoma (FL) is an indolent, but incurable form of non-Hodgkin lymphoma (NHL), with variable clinical behavior. The immune environment plays a major role in dictating the biology of FL. For example, gene expression profiling experiments have demonstrated that recurring “immune response” gene signatures in diagnostic FL specimens were more accurate in predicting clinical outcomes than gene expression patterns in FL cells themselves. Furthermore, immune cells, including T cells, are often found infiltrating or surrounding malignant follicles in FL, and our group has identified the presence of highly clonal T cell populations in most FL samples, suggesting that FL cells express antigens that drive the expansion of lymphoma-specific T cells. In addition, inactivating mutations in B2 microglobulin are relatively common in FL, indicating that down-regulation of antigen expression may occur as a result of host immune surveillance. Lastly, FL is responsive to immunotherapies, such as PD-1 antibody blockade. Importantly, the FL antigens that promote spontaneous immune recognition remain unknown. Emerging data has revealed a critical relationship between the genetic landscape and the immunogenicity of cancer. Somatic mutations in malignant cells encode for altered proteins (i.e. neoantigens) that are “sensed” as foreign by T cells, and the mutational “load” of a given tumor has direct implications for the host immune response.Methods: To address the hypothesis that somatic mutations generate neo-antigens which are the targets of T cell recognition in patients with FL, whole exome sequencing (WES) was performed on FACS-purified FL B cells (CD10+CD19+ cells) and matched T cells from 7 FL biopsy samples. HLA A, B and C haplotypes were predicted using PolySolver, OptiType, Phlat and Seq2hla software with >90% concordance. HLA binding affinity was predicted for all possible 8-11-mer peptides generated from each somatic mutation and corresponding wild-type peptides using NetMHCpan v2.8. These tiled peptides were analyzed for their predicted binding affinities (IC50 nM) to HLA class I molecules. An IC50 <150 nM was considered a predicted strong binder, ≥150 but ≤500 nM an intermediate to weak binder, and >500 nM a non-binder.Results: A median number of 71 somatic mutations were identified in each sample (range, 8-135). Non-synonymous single nucleotide variants (SNV) were cataloged, and in silico analysis was utilized to predict potential high-affinity epitopes capable of binding patient-specific HLA molecules. We achieved an average of 90% proportion of nucleotides > Phred quality score 30 of total bases, according to the base calling accuracy (range, 89.02%-92.06%). G>A/C>T transitions were the most common nucleotide substitutions observed. Consistent with published reports, recurrent somatic mutations were identified in EZH2, CREBBP, IGLL5, MUC21, PCLO, and PIM1. We also identified recurrent mutations in BCL2 and MLL2 (frameshift insertions) known to dominate the mutational landscape of FL. Mutations in HLA class II alleles were common, occurring in 3/7 samples. In silico analysis was next employed to identify putative neo-antigens. A total of 139 candidate neo-antigenic peptides were predicted across all patients. On average, 19 neo-antigens were predicted for each patient (range, 3-41). No public neo-antigens were identified, suggesting that the neo-antigens arising in FL are patient-specific. This observation is consistent with what has been described for other cancer subtypes. Functional validation of lead candidate neo-antigen peptides is ongoing.Conclusion: While overall numbers of somatic mutations are modest in FL, putative neo-antigens were identified in all patient samples analyzed. Our findings suggest that immune therapies incorporating neo-antigen-specific approaches, such as personalized vaccines, should be explored in FL patients. DisclosuresNo relevant conflicts of interest to declare.

Targeted Treatment of Follicular Lymphoma.

Follicular lymphoma (FL) is the most common indolent B-cell lymphoma. Advanced stage disease is considered incurable and is characterized by a prolonged relapsing/remitting course. A significant minority have less favorable outcomes, particularly those with transformed or early progressive disease. Recent advances in our understanding of the unique genetic and immune biology of FL have led to increasingly potent and precise novel targeted agents, suggesting that a chemotherapy-future may one day be attainable. The current pipeline of new therapeutics is unprecedented. Particularly exciting is that many agents have non-overlapping modes of action, offering potential new combinatorial options and synergies. This review provides up-to-date clinical and mechanistic data on these new therapeutics. Ongoing dedicated attention to basic, translational and clinical research will provide further clarity as to when and how to best use these agents, to improve efficacy without eliciting unnecessary toxicity.

EZH2 Expression in Follicular Lymphoma Is Variable and Independent from the Progression of Disease Within 24 Months of First Treatment.

Follicular lymphoma (FL) relapse within 24 months of the first immunochemotherapy (POD24) indicates more precisely poor overall survival and high risk of death. The aim of the study was to assess the potential value of POD24 in FL and describe the enhancer of zeste homolog 2 (EZH2) expression profile, in correlation with clinical/histopathological/immunophenotypical characteristics. This retrospective single-center study included 75 patients with FL treated under watch and wait (W&W) and immunochemotherapy regimens. All cases were immunohistochemically assessed: assays were performed for EZH2, CD10, BCL6, BCL2, MUM1, MYC and p53. POD24 was independent of clinical/histopathological/immunohistochemical features and separated patients with inferior outcomes. EZH2 high expression was observed in high/low grade and follicular/diffuse FL patterns. BCL2-negative (p=0.042) and MUM1 (p=0.039), MYC (p<0.001), p53 (p<0.001) - positive cases had significantly higher EZH2 expression. POD24 is currently the most useful tool for the identification of poor outlook patients. EZH2 is crucial in FL biology, but the value of its protein expression is limited as a prognostic factor.

Integration and Iteration: Using Advanced, High-Content Imaging and Single-Cell Gene Expression Analysis to Uncover Unique Aspects of Follicular Lymphoma Biology

BACKGROUND: Follicular lymphoma (FL) is an indolent malignancy of germinal center B-cell origin. FL patients experience remarkable heterogeneity in their disease trajectory, with many patients slowly progressing over several years, and a subset of patients experiencing an aggressive clinical course. Uncovering the cell-intrinsic and -extrinsic factors that govern differential progression and outcome in FL patients is thus essential. Beyond the genetic and epigenetic aberrations that contribute to FL oncogenesis, the tumor microenvironment (TME) plays an integral role in supporting the proliferation and survival of malignant cells. In a process described as "re-education", FL tumor cells may actively subvert the normal functions of non-malignant cells present in the TME, including T cells, follicular dendritic cells (FDCs), macrophages, dendritic cells, and stromal cells to support their survival and growth. METHODS: To address the importance of the TME in FL, we molecularly profiled excisional lymph node biopies from untreated FL patients using multiple platforms: bulk RNA sequencing (RNAseq), single-cell RNA sequencing (scRS), and a unique high content imaging method, Iterative Bleaching Extends MultipleXity (IBEX), which utilizes chemical bleaching to image 40+ proteins in the same tissue section by antibody staining. In combination with advanced computational tools for the quantitative analysis of cell types and distribution in tissues, we have used this approach to evaluate the TME:FL interaction within 8 FL samples, with 4 normal lymph nodes as controls. RESULTS: Both FL and TME components reconstructed from bulk RNA-seq were similar to the cellular composition revealed by scRS and IBEX analyses. Moreover, the bulk RNAseq and scRS identified the expression of genes involved in tumorigenesis and oncogenic signaling, often unique to each case. However, RNAseq-based approaches often miss important cellular and acellular components not readily extracted from dense tissues. For example, IBEX imaging can trace the pattern of blood vessels within a section, which cannot be achieved with non-imaging methods. In one case, our multi-parameter imaging studies revealed the close spatial interaction between clonal FL B cells, expressing a B-cell receptor (BCR) possessing a de novo N-linked glycosylation site introduced by somatic hypermutation, and cells expressing dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). This contact may activate pro-survival signaling in the malignant B cells. CONCLUSIONS: Integration of bulk RNAseq, scRS, clonotype analysis, and IBEX reveals both shared and unique aspects of different FL tumors. These data highlight the importance of integrating direct tissue analysis by high-content imaging with methods examining aspects of isolated cells. This approach may provide a more complete understanding of tumor biology, which in turn will identify patients at risk for developing aggressive disease and rationally improve treatment strategies for FL. This research was supported in part by the Intramural Research Program of the NIH, NIAID and NCI Figure Disclosures Bagaev: BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Plotnikova:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Galkin:BostonGene: Current Employment, Patents &amp; Royalties. Postovalova:BostonGene: Current Employment, Current equity holder in private company. Svekolkin:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Isaev:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Lozinsky:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Meerson:BostonGene: Current Employment. Varlamova:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Ovcharov:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Polyakova:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Nomie:BostonGene: Current Employment, Current equity holder in private company. Kotlov:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Tsiper:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Frenkel:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Attaulakhanov:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties. Fowler:BostonGene: Current Employment, Current equity holder in private company, Patents &amp; Royalties.

Site to Site Comparison of Follicular Lymphoma Biopsies By Single Cell RNA Sequencing

Site to Site Comparison of Follicular Lymphoma Biopsies By Single Cell RNA Sequencing

Progression of Disease Within 24 Months in Follicular Lymphoma Is Associated With Reduced Intratumoral Immune Infiltration.

PURPOSEUnderstanding the immunobiology of the 15% to 30% of patients with follicular lymphoma (FL) who experience progression of disease within 24 months (POD24) remains a priority. Solid tumors with low levels of intratumoral immune infiltration have inferior outcomes. It is unknown whether a similar relationship exists between POD24 in FL.PATIENTS AND METHODSDigital gene expression using a custom code set—five immune effector, six immune checkpoint, one macrophage molecules—was applied to a discovery cohort of patients with early- and advanced-stage FL (n = 132). T-cell receptor repertoire analysis, flow cytometry, multispectral immunofluorescence, and next-generation sequencing were performed. The immune infiltration profile was validated in two independent cohorts of patients with advanced-stage FL requiring systemic treatment (n = 138, rituximab plus cyclophosphamide, vincristine, prednisone; n = 45, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone), with the latter selected to permit comparison of patients experiencing a POD24 event with those having no progression at 5 years or more.RESULTSImmune molecules showed distinct clustering, characterized by either high or low expression regardless of categorization as an immune effector, immune checkpoint, or macrophage molecule. Low programmed death-ligand 2 (PD-L2) was the most sensitive/specific marker to segregate patients with adverse outcomes; therefore, PD-L2 expression was chosen to distinguish immune infiltrationHI (ie, high PD-L2) FL biopsies from immune infiltrationLO (ie, low PD-L2) tumors. Immune infiltrationHI tissues were highly infiltrated with macrophages and expanded populations of T-cell clones. Of note, the immune infiltrationLO subset of patients with FL was enriched for POD24 events (odds ratio [OR], 4.32; c-statistic, 0.81; P = .001), validated in the independent cohorts (rituximab plus cyclophosphamide, vincristine, prednisone: OR, 2.95; c-statistic, 0.75; P = .011; and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone: OR, 7.09; c-statistic, 0.88; P = .011). Mutations were equally proportioned across tissues, which indicated that degree of immune infiltration is capturing aspects of FL biology distinct from its mutational profile.CONCLUSIONAssessment of immune-infiltration by PD-L2 expression is a promising tool with which to help identify patients who are at risk for POD24.

Novel Findings in Follicular Lymphoma Pathogenesis and the Concepts of Targeted Therapy

The molecular pathogenesis of follicular lymphoma (FL) was partially revealed by the discovery of BCL2 translocations to the region encoding the immunoglobulin heavy chain, which accompany the vast majority of cases. This aberration leads to the ectopic and constitutive expression of anti-apoptotic BCL2 protein in B-cells. Nevertheless, the aberration alone is not sufficient for FL development, which suggests necessity of further genetic aberrations acquisition for neoplastic transformation to FL. Their discovery has been enabled by recent progress in the field of massive parallel sequencing (next generation sequencing), which revealed high number of genetic aberrations connected with onset and progression of FL. The occurrence of many of these aberrations in the early stages of the disease, and the fact that they are shared by the majority of patients with FL, fundamentally changed our former understanding of the disease onset. Furthermore, in a large fraction of patients, FL undergoes histological transformation to a more aggressive lymphoma, which is also associated with specific genetic alterations. In this review, we summarize the current knowledge of molecular pathways connected with FL biology and discuss their role in the context of normal B-cell development. Understanding of FL biology is essential for the development of new targeted therapies and the stratification of patients, and potentially also for the selection of treatment for specific patients who share the same genetic aberrations.Key words: follicular lymphoma - mutation - aberration - apoptosis - epigenetic regulators - microRNA This research was carried out under the project CEITEC 2020 (LQ1601) with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustain ability Programme II. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 28. 1. 2017Accepted: 5. 3. 2017.

Follicular lymphoma: State-of-the-art ICML workshop in Lugano 2015.

The 13th International Conference on Malignant Lymphoma held in Lugano in June 2015 was preceded by a closed workshop (organized in collaboration with the American Association for Cancer Research and the European School of Oncology) with the aim of developing an up-to-date understanding of the biology of follicular lymphoma and the clinical implications of new findings in the field. Discussed topics included the mutational spectrum at diagnosis, the clinical correlates of genetic and epigenetic alterations, the mechanisms of clonal evolution and histological transformation, the cross talk between tumor cells and microenvironment, and the development of novel treatments. This report represents a summary of the workshop.