Pathogenesis Of Follicular Lymphoma Research Articles

Causal relationship between circulating cytokines and follicular lymphoma: a two-sample Mendelian randomization study.

Follicular lymphoma (FL), derived from germinal centre (GC) B cells, is a kind of systemic neoplasm. Even though FL is indolent, it remains an incurable haematology Neoplasm. Accumulating evidence has suggested that the circulating cytokine is associated with the development of FL, yet the causal relationship between FL and circulating cytokines remains undetermined. Therefore, we conducted a two-sample Mendelian randomization (MR) to confirm the causal link between FL and levels of circulating cytokines with the use of summary data on circulating cytokines and FL. All these data from genome-wide association study were derived from the Genome-wide pQTL mapping which contains 14,824 individuals. FL data were acquired exclusively from FinnGen, where 218,792 individuals (522 cases vs. 218,270 controls) were involved. Various statistical methods, including the inverse variance weighted method (IVW), weighted median (WME), simple model, weighted model (WM) and MR-Egger, were used to evaluate the potential causal connection between circulating cytokines and FL. Sensitivity analysis, which involves the examination of the heterogeneity, pleiotropy, and leave-one-out method, was also performed to ensure more trustworthy results. A bidirectional MR test was performed to evaluate the direction of causal association between circulating cytokines and FL. Combining all the steps of MR analysis, we revealed four causal cytokines: C-X-C motif chemokine ligand 5 (CXCL5), interleukin-15 receptor A (IL15RA), interleukin-20 (IL20), and neurotrophin-3 (NT-3). The risk of FL may be inversely linked to CXCL5 (OR=0.73, CI: 0.545-0.979, P=0.036), IL-15RA (OR=0.669, CI: 0.451-0.993, P=0.046), and IL-20 (OR=0.565, CI: 0.325-0.981, P=0.043) but positively linked to NT-3 (OR=1.872, CI: 1.063-3.297, P=0.03). In addition, in our study, no causal effect of FL on cytokines was demonstrated and no significant heterogeneity and pleiotropy were found. Our research revealed the causal relationship between cytokines and FL, along with both the anti-protective effect of CXCL5, IL-15RA, and IL-20 and the protective effect of neurotrophin-3 on FL. These findings aim to provide new clues regarding the pathogenesis of FL and to extend the potential of circulating cytokines to therapeutic interventions.

Gene targeted and immune therapies for nodal and gastrointestinal follicular lymphomas.

Follicular lymphoma (FL) is the most common indolent B-cell lymphoma (BCL) globally. Recently, its incidence has increased in Europe, the United States, and Asia, with the number of gastrointestinal FL cases expected to increase. Genetic abnormalities related to t(14;18) translocation, BCL2 overexpression, NF-κB pathway-related factors, histone acetylases, and histone methyltransferases cause FL and enhance its proliferation. Meanwhile, microRNAs are commonly used in diagnosing FL and predicting patient prognosis. Many clinical trials on novel therapeutics targeting these genetic abnormalities and immunomodulatory mechanisms have been conducted, resulting in a marked improvement in therapeutic outcomes for FL. Although developing these innovative therapeutic agents targeting specific genetic mutations and immune pathways has provided hope for curative options, FL treatment has become more complex, requiring combinatorial therapeutic regimens. However, optimal treatment combinations have not yet been achieved, highlighting the importance of a complete under-standing regarding the pathogenesis of gastrointestinal FL. Accordingly, this article reviews key research on the molecular pathogenesis of nodal FL and novel therapies targeting the causative genetic mutations. Moreover, the results of clinical trials are summarized, with a particular focus on treating nodal and gastrointestinal FLs.

Localization of DC-SIGN Expressing Cells in Follicular Lymphoma

The high incidence of unusual mannose-rich glycans on the expressed surface immunoglobulins of follicular lymphoma (FL) suggests a pathogenic role for this modification. While lectin-mediated signaling has been proposed to provide trophic support to FL cells, the localization of the lectins has not been extensively characterized. Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) is a mannose-selective lectin named for its expression on classic marrow-derived dendritic cells. In vitro, recombinant DC-SIGN induces signaling in primary FL cells. The nodules of Follicular lymphoma (nFL) are the neoplastic counterparts of normal germinal centers (GC). Both GC and nFL contain variable numbers of centrocytic and/or centroblastic B cells, T-cells, and follicular dendritic cells (FDCs), but macrophages are typically found only in GC. Although termed “follicular dendritic cells”, FDCs are thought to be derived from non-blood stem-cells, potentially pericytes. Therefore, nFL lack classic dendritic cells and macrophages so it is unclear what cells in FL could express DC-SIGN. Therefore, we systematically characterized FDC and DC- SIGN localization in sixteen FL and nine Follicular hyperplasia (FH) lymph nodes. Using flow cytometry of disaggregated FL nodes, we were not able to identify a specific cell population expressing DC-SIGN. However, using immunohistochemistry and Immunofluorescence methods, we identified nine cases of FH and five cases of FL (n=5/16) with DC-SIGN and FDC co-localization. Immunohistochemical stains of the FDCs using CD23 and CD21 showed that the follicular dendritic structures were disrupted in almost all the FL cases assessed showing either a peripheral crescentic staining or broken FDC meshwork within the follicle. We identified five cases of FL with an average of more than four well visualized and complete FDC structures staining per low power field (4X) while the remaining cases had an average of less than two complete FDC network staining. There was near complete absence of CD21 positive FDCs meshwork identified in 50% of the FL cases compared to only one case lacking CD23 positive FDCs structure staining. In contrast, FH cases (n=9) showed complete staining of the FDC meshwork within the follicles with both CD21/CD23. For FH, the average number of complete FDC staining with CD23 was 18 while an average of 10 complete FDC meshworks stained with CD21. Furthermore, immunofluorescence imaging analysis showed different pattern of localization of CD23 positive FDC with DC-SIGN with between the two conditions. In FH, DC-SIGN and CD23 colocalization was identified mainly in the light zone. For FL, we observed that DC-SIGN overlap with CD23 localized in the intra-follicular areas and co-localized with CD163 (M2 macrophages) in the inter-follicular zones. Confocal imaging confirmed colocalization of CD23 and DC-SIGN on FDCs with close association to MUM1-positive B cells in both GC and nFL. Additional analysis also showed that 80% (n=5) of the cases which had more than four complete FDC structures staining with CD23 co-localized with DC-SIGN in the intra-follicular areas and the cases with complete FDC network disruption did not. There was no significant relationship identified between the extent of FDC networks and the fraction of non-neoplastic B cells in the FL cases. In summary, our data show that DC-SIGN is expressed on FDCs in FL and FH even though FDCs are not the marrow-derived DCs for which DC-SIGN is named. Furthermore, localization of DC-SIGN to the follicle suggests a potential role for the microenvironment in the pathogenesis of FL and possible therapeutic target.

Co-Occurrence of Clonally Related Follicular Lymphoma and Histiocytic Sarcoma

Histiocytic sarcoma (HS) is an extremely rare and aggressive malignancy thought to be derived from mononuclear phagocytic cells. HS may occur as primary tumor or may be clonally related to a hematologic malignancy. Rare cases of HS have been reported in patients with follicular lymphoma (FL). Prior reports have demonstrated a common clonal origin of FL and HS in many cases via demonstration of identical BCL2/IGH [t(14;18)] translocations in both malignancies. As this is likely the earliest event in FL pathogenesis, the mechanisms by which HL may arise from FL remain unknown. A greater understanding of how a B cell malignancy can transdifferentiate into a myeloid one would yield insights into the fundamental underlying biology. From a patient with long-standing history of FL and subsequent diagnosis of HS, we collected tumor biopsies from a tumor site harboring both FL and HS. Tumor biopsies were subjected to standard histopathological assays, as well as targeted hybrid-capture DNA sequencing of 164 genes commonly mutated in hematologic malignancies and cytogenetic assays for the BCL2 rearrangement (Figure 1). Additionally, we performed single cell RNA sequencing (scRNA-seq) using the 10X chromium followed by single cell long read sequencing using Nanopore Oxford Technologies (Figure 1). Two tumor populations were evident in the scRNAseq data - FL tumor cells, identified by homogeneity of BCR light chain variable gene expression and expression of FL marker genes, and a separate cluster of cells expressing CD68, CD163 and S100A8, characteristic for HS cells. Gene expression profiles of the two tumor populations revealed high expression of the targetable “don´t eat me” signal CD47 on both populations. HS also expressed BCL2, hinting at a common clonal origin of FL and HS neoplasms. Indeed, fluorescent in situ hybridization (FISH) studies demonstrated the presence of t(14;18) in both the FL and HS tumor cells. To understand if similar transcription factors (TFs) were active in the two malignancies, we inferred the top 10 enriched TF for each tumor subpopulation and found that while TFs like IRF8 and REL were enriched in FL cells, HS cells showed a higher expression of MYC, STAT2 and CEBPD. Bulk targeted hybrid-capture DNA sequencing of the tumor sample revealed mutations in genes frequently altered in FL ( e.g.CREBBP, BIRC3, and PIM1) as well as in a gene commonly mutated in HS ( MAP2K1). To gain deeper insight into the clonal relationship between the two malignancies within the same tumor, we conducted long read sequencing on the barcoded single cell-cDNA. We are currently analyzing the data in two ways. First, we are searching for the mutations identified in bulk sequencing now in the single cell long read data from the FL and HS cells, using microenvironmental immune cells from the same tumor as a negative control. We have confirmed that these genes are all expressed in the FL, HS, and immune cells, providing transcripts for us to analyze for mutations. Second, we are employing an unbiased approach for identifying genetic variants, again comparing FL and HS cells. This data will be available by the annual meeting. By applying single cell genomics analyses to a combined FL/HS biopsy, our study aims to understand the evolutionary relationship of HS arising from FL, particularly whether the HS arose from an early dormant clone (branched evolution) or from a late, prevalent Fl clone (linear evolution). Evidence of the former would strengthen the view that common progenitor cells (CPCs) in FL are capable of differentiating into myeloid and lymphoid malignancies.

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.

Novel insights into the pathogenesis of follicular lymphoma by molecular profiling of localized and systemic disease forms

Knowledge on the pathogenesis of FL is mainly based on data derived from advanced/systemic stages of FL (sFL) and only small cohorts of localized FL (lFL) have been characterized intensively so far. Comprehensive analysis with profiling of somatic copy number alterations (SCNA) and whole exome sequencing (WES) was performed in 147 lFL and 122 sFL. Putative targets were analyzed for gene and protein expression. Overall, lFL and sFL, as well as BCL2 translocation-positive (BCL2+) and –negative (BCL2−) FL showed overlapping features in SCNA and mutational profiles. Significant differences between lFL and sFL, however, were detected for SCNA frequencies, e.g., in 18q-gains (14% lFL vs. 36% sFL; p = 0.0003). Although rare in lFL, gains in 18q21 were associated with inferior progression-free survival (PFS). The mutational landscape of lFL and sFL included typical genetic lesions. However, ARID1A mutations were significantly more often detected in sFL (29%) compared to lFL (6%, p = 0.0001). In BCL2 + FL mutations in KMT2D, BCL2, ABL2, IGLL5 and ARID1A were enriched, while STAT6 mutations more frequently occurred in BCL2- FL. Although the landscape of lFL and sFL showed overlapping features, molecular profiling revealed novel insights and identified gains in 18q21 as prognostic marker in lFL.

Use of obinutuzumab in therapy of patients with newly diagnosed follicular lymphoma. Experience of the Oncohematology Division of the Clinical Hospital “Lapino”

Follicular lymphoma (FL) is the most common indolent form of lymphoma and characterized by recurrent course and heterogenous clinical signs. Selection of treatment program for patients with newly diagnosed or recurrent FL is based primarily on the size of the tumor. While use of rituximab-containing regimens led to increased progression-free survival, in some cases tumors are refractory to rituximab. Understanding of molecular mechanisms of FL pathogenesis and action of anti-CD20 monoclonal antibodies allowed to develop new drugs with several advantages compared to rituximab. According to the data of large randomized trials, use of glycoengineered monoclonal anti-CD20 type II antibody obinutuzumab in combination with chemotherapy in patients with FL has manageable toxicity profile and longer progression-free survival and event-free survival compared to use of rituximab.

Multi-Omics Profiling of Longitudinal Samples Reveals Early Genomic Changes in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) is an indolent malignancy, characterized by multiple relapses during the disease course. Annually around 2-3% of patients experience transformation to aggressive disease (tFL), most commonly to diffuse large B-cell lymphoma (DLBCL). Transformation and progression of disease within 2 years (POD24) are associated with poor prognosis, yet the molecular events underlying these processes are not well understood. The existence of common progenitor cells (CPCs) has been inferred from genetic analyses of longitudinal biopsies. Improved characterization of genetic alterations associated with CPCs in cases with transformation and POD24 may improve our understanding of disease progression, and reveal molecular markers for high-risk disease. Methods: To identify early genetic events during FL pathogenesis and molecular events underlying progression and transformation, we performed whole-exome sequencing of 97 serial tumor biopsies and matched normal samples purified from peripheral blood from 44 FL patients; 22 who experienced transformation (tFL) and 22 with progression of FL (nFL). Nineteen patients(both groups) experienced POD24. SNP6.0 data was available for 93 sequenced tumors and was used to infer allele-specific copy number alterations. Bulk RNA sequencing data was available for 64 sequenced tumors. Several computational tools were applied to identify potential cancer driver genes (IntOGen, MutSig2CV, 2020plus), mutational signatures, and to study clonal evolution (PyClone, ClonEvol). CIBERSORTx was used to infer B-cell specific transcriptomes from bulk RNAseq data. Results: Deep whole exome sequencing confirmed recurrent mutations in epigenetic regulators (CREBBP, KMT2D, EZH2, and EP300) and known copy number alterations. The mutational landscape was similar in nFL and tFL patients, except for higher recurrence of EZH2 mutations in tFL. We used CIBERSORTx to impute B-cell transcriptomes from bulk RNA-sequencing data to identify differentially expressed genes associated with common mutations. We discovered that numerous histone genes were downregulated in biopsies with EZH2 mutations, and in pretreatment samples from tFL patients, potentially contributing to epigenetic deregulation. Based on the mutational pattern in each biopsy, we calculated the genomic distances between longitudinal samples. This revealed great variability in nFL and tFL patients, compatible with complex evolutionary patterns independent of transformation. Tumor clonal evolution analysis identified CREBBP and KMT2D mutations as early genetic events in FL pathogenesis, likely occurring prior to the early CNAs that remained stable throughout disease progression such as gain in 12p and 18p and loss in 6p. Conclusions: Identification of such early and potentially essential genetic events may therefore provide opportunities for early disease detection and monitoring of progression.

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

Molecular Pathogenesis of Follicular Lymphoma: From Genetics to Clinical Practice

Follicular lymphoma (FL), a generally indolent disease that derives from germinal center (GC) B cells, represents around 20–25% of all new lymphomas diagnosed in Western countries. The characteristic t(14;18)(q32;q21) translocation that places the BCL2 oncogene under control of the immunoglobulin heavy-chain enhancer occurs in pro- or pre-B cells. However, additional secondary alterations are required for the development of overt FL, which mainly affects genes involved in epigenetic and transcriptional regulation, signaling and B cell differentiation, the BCR/NF-κB pathway, and proliferation/apoptosis. On the other hand, new insights into the FL pathogenesis suggest that FL lacking the BCL2 translocation might be a distinct biological entity with genomic features different from the classical FL. Although FL is considered an indolent disease, around 10–20% of cases eventually transform to an aggressive lymphoma, usually a diffuse large B cell lymphoma, generally by a divergent evolution process from a common altered precursor cell acquiring genomic alterations involved in the cell cycle and DNA damage responses. Importantly, FL tumor cells require interaction with the microenvironment, which sustains cell survival and proliferation. Although the use of rituximab has improved the outlook of most FL patients, further genomic studies are needed to identify those of high risk who can benefit from innovative therapies. This review provides an updated synopsis of FL, including the molecular and cellular pathogenesis, key events of transformation, and targeted treatments.

Identification of Hub Genes and Key Pathways Associated with Follicular Lymphoma

Follicular lymphoma (FL) is the second most prevalent form of non-Hodgkin lymphoma (NHL) and accounts for almost 20% of all NHL cases. Although FL patients' overall survival rates have steadily increased, there is still no accepted standard of care for individuals who experience recurrence or resistance to treatment. Hence, it is needed to evaluate the precise molecular cascades underlying FL to develop efficient diagnostic and treatment approaches. Herein, we aimed to evaluate variations in gene expression profiles, explore the underlying mechanisms, and find new FL targets. In the present study, Gene Expression Omnibus (GEO) database was employed to evaluate microarray datasets including GSE32018 and GSE55267. R software was employed to evaluate differentially expressed genes (DEGs) between FL and noncancer samples. The DEGs were evaluated using GO, KEGG pathway enrichment analysis, and PPI network to evaluate hub genes, which were then, examined using gene function enrichment analysis. According to the obtained results, a total of 190 upregulated and 162 downregulated DEGs were evaluated. Following the generation of PPI networks, 15 hub genes in highly connected upregulated DEGs were selected including FN1, MMP9, CCL2, CD8A, POSTN, CCR5, COL3A1, CXCL12, VCAM1, COL1A2, CCL5, SPARC, TIMP1, CXCL9, and IL18. The GO enrichment evaluation of the underlined hub genes indicated that the immunological response was the most considerably enriched term. Twelve significant cascades were found using the KEGG pathway analysis, most of which were linked to cellular structure and immunity. Our findings suggested that FN1, SPARC, POSTN, MMP9, and VCAM1 genes are potential biomarkers of FL, and cellular immunity contributes to the pathogenesis of FL. Moreover, the unique DEGs and cascades found in the present study may present new perspectives on the molecular basis of FL's underlying mechanisms as well as a new understanding of FL's future precise management.

Plasma Concentrations and Cancer-Associated Mutations in Cell-Free Circulating DNA of Treatment-Naive Follicular Lymphoma for Improved Non-Invasive Diagnosis and Prognosis.

Follicular lymphoma (FL) is the second most frequent non-Hodgkin lymphoma accounting for 10-20% of all lymphomas in western countries. As a clinically heterogeneous cancer, FL occasionally undergoes histological transformation to more aggressive B cell lymphoma types that are associated with poor prognosis. Here we evaluated the potential of circulating cell-free DNA (cfDNA) to improve the diagnosis and prognosis of follicular lymphoma patients. Twenty well-characterized FL cases (13 symptomatic and 7 asymptomatic) were prospectively included in this study. Plasma cfDNA, formalin-fixed paraffin-embedded (FFPE) tumor tissue DNA, and patient-matched granulocyte genomic DNA samples were obtained from 20 treatment-naive FL cases. Ultra-deep targeted next-generation sequencing was performed with these DNA samples by using a custom-designed platform including exons and exon-intron boundaries of 110 FL related genes. Using a strict computational bioinformatics pipeline, we identified 91 somatic variants in 31 genes in treatment-naive FL cases. Selected variants were cross-validated by using PCR-Sanger sequencing. We observed higher concentrations of cfDNA and a higher overlap of somatic variants present both in cfDNA and tumor tissue DNA in symptomatic FL cases compared to asymptomatic ones. Variants known to be associated with FL pathogenesis such as STAT6 p.D419 or EZH2 p.Y646 were observed in patient-matched cfDNA and tumor tissue samples. Consistent with previous observations, high Ki-67 staining, elevated LDH levels, FDG PET/CT positivity were associated with poor survival. High plasma cfDNA concentrations or the presence of BCL2 mutations in cfDNA showed significant association with poor survival in treatment-naive patients. BCL2 mutation evaluations in cfDNA improved the prognostic utility of previously established variables. In addition, we observed that a FL patient who had progressive disease contained histological transformation-associated gene (i.e. B2M and BTG1) mutations only in cfDNA. Pre-treatment concentrations and genotype of plasma cfDNA may be used as a liquid biopsy to improve diagnosis, risk stratification, and prediction of histological transformation. Targeted therapies related to oncogenic mutations may be applied based on cfDNA genotyping results. However, the results of this study need to be validated in a larger cohort of FL patients as the analyses conducted in this study have an exploratory nature.

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.

Epigenetic modification as a therapeutic approach for B-cell lymphoma

Genes that regulate immunological activities are transiently suppressed by epigenetic modification during the germinal center reaction of B cells and reactivated when B cells exit the germinal center. Mutations of EZH2 and other epigenetic modifier genes are frequently involved in the pathogenesis of follicular lymphoma and lead to silencing of the genes necessary for exiting the germinal center. Tazemetostat, an EZH2 inhibitor, has been approved for the treatment of follicular lymphoma with EZH2 gain-of-function mutations in Japan. Tazemetostat restores the expressions of MHC and CD58 in lymphoma cells and synergistically enhances the immune reactions of T and natural killer cells against lymphoma cells. Tazemetostat also induces lymphoma cells to secrete CCL17/TARC and enhances T-cell migration. CD58 and CCL17 are known to play central roles in the formation of T-cell-rich tumor microenvironment of Hodgkin lymphoma. We found that tazemetostat enhances the expression of genes overexpressed in Hodgkin/Reed-Sternberg cells. Epigenetic modifiers and new molecular targeted therapies are expected to provide new insights into the pathogenesis of lymphoma and mechanisms determining the histology of lymphoma.

M6A Modification Patterns Identify a Subset of Follicular Lymphoma Harboring an Exhausted Tumor Microenvironment

Background: Epigenetic dysregulation is important for the pathogenesis of follicular lymphoma (FL). N6-methyladenosine (m6A) is the most common post-transcriptional modification of mRNA. The m6A modification affects a wide range of cellular processes, including RNA stability, export, splicing or translation, and is extensively involved in the development and progression of multiple non-hematological and hematological malignancies. However, the role of m6A modification in FL remain largely unknown.Methods: We comprehensively evaluated the m6A modification patterns of 351 FL patients from three cohorts based on 16 m6A regulators and constructed the m6A gene signature the by unsupervised clustering analysis. The m6A score was then established using principal component analysis. The association between m6A modification patterns and prognostic implications, biological processes, and microenvironment characterizations in FL were further investigated.Results: A total of 16 m6A regulators were finally identified in this study, including 8 writers, 7 readers and one eraser. Two distinct m6A modification patterns, termed as m6A cluster A and m6A cluster B respectively, were identified based on the similarity displayed by the expression levels of 16 m6A regulators and the proportion of ambiguous clustering measure. Patients with m6A cluster A had significantly poorer survival than those with m6A cluster B (p=0.0085) and showed enrichment in cell cycle, DNA replication and mismatch repair biological processes. However, patients with m6A cluster B were markedly enriched in MAPK pathway, WNT pathway, mTOR pathway, natural killer cell mediated cototoxicity, cytokine-cytokine receptor interaction and T-cell receptor pathway. Additionally, myeloid dendritic cell and CD8 + T cell were significantly enriched in m6A cluster A, nevertheless CD4 + T cell was abundant in m6A cluster B. Base on the DEGs between m6A cluster A and m6A cluster B, a m6A gene signature was further constructed, namely m6A gene signature A and m6A gene signature B. Patients with m6A gene signature B experienced worse outcome than those with m6A gene signature A. Simultaneously, a m6A scoring model was also established to quantify the m6A modification pattern of individual patients with FL. Patients with low m6A score presented a prominent inferior survival than those with high m6A score (p< 0.0001). The median survival times for patients with low and high m6A score were 8.84 (95% confidence interval [CI]: 7.251-10.429) and 15.73 years (95% CI: 11.729-19.731), respectively. High m6A score group was remarkably riched in macrophage cell and CD4 + T cell infiltration, while low m6A score group exhibited significantly increased myeloid dendritic cell and CD8 + T cell infiltration, suggesting that m6A score could distinguish populations with distinct immune cell infiltration characteristics. GSVA analysis showed that the expression of genes involved in the immune and inflammatory response was significantly upregulated in low m6A score group. But we finally found that the CD8 + T cells in low m6A score group kept in a state of exhaustion, leading to that exhausted T cells were enriched in low m6A score group. The expression of PD-1 and PD-L1 in low m6A score group was also higher than those in high m6A score group. GSEA analysis showed that low m6A score group exhibited an enhanced IFN-γ response. Finally, we verified the value of the m6A score for predicting response to anti-PD-L1 antibody in external immunotherapy cohort (IMvigor210), and found that patients with low m6A score exhibited significantly clinical benefits and a markedly prolonged survival from anti-PD-L1 therapy.Conclusions: This is the first time to comprehensively investigate the role of m6A modification in FL. The m6A score identifies a subset of follicular lymphoma harboring an exhausted tumor microenvironment and helps for the selection of patients for immunotherapy, guiding the personalized immunotherapy in FL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tumor microenvironment of follicular lymphoma

: Follicular lymphoma (FL) is the second most common type of non-Hodgkin lymphoma and accounts for approximately 20% of all new non-Hodgkin lymphomas in western countries. Although FL generally has an indolent natural history, a subset can transform into high-grade lymphomas such as diffuse large B-cell lymphoma (DLBCL) or progress rapidly with associated poor clinical outcomes. The development of more effective means of FL control prior to transformation or progression is likely to be key to improving results. Over the past 10–15 years there has been a rapid accumulation in studies focusing on the FL tumor microenvironment, leading to an expansion in our understanding of this complex and complicated neighbourhood. The FL microenvironment is distinct from other B cell lymphomas, with a complex interplay of cellular and non-cellular components, and plays a critical role in the pathogenesis of this disease. As part of this special series on FL, this Review covers our current knowledge of the FL tumor microenvironment with a focus on the cellular composition, key genetic aberrations, disturbances in the cytokine milieu and the neoantigen landscape of FL. We then explore the impact of the FL tumor microenvironment on risk stratification strategies and role of novel therapeutics targeting the microenvironment. Given the critical role of the microenvironment in the pathogenesis of FL, and the exciting possibility of novel therapeutic avenues, the objective of this review is to provide the reader with a summary to understand the key literature and highlight gaps in knowledge that should be prioritised in the pursuit of more effective therapies.

Molecular Pathogenesis of Follicular Lymphoma and Its Relevance to Clinical Practice

Introduction Our understanding of the molecular basis of follicular lymphoma (FL) has come a long way since the identification of the reciprocal chromosomal translocation, t(14;18), heralded as the primary genetic hallmark.1 Over thirty years on, we are approaching a near-complete catalogue of the genetic underpinnings of this lymphoma subtype, primarily ushered in by technological advancements, such as next-generation sequencing. What is apparent from recent studies is that most FL tumours harbour t(14;18) together with additional genetic alterations, affecting numerous biological pathways, particularly genes involved in epigenetic regulation.2 Moreover, the relapsing-remitting pattern of FL has provided the opportunity to trace the life history and patterns of genetic heterogeneity within an individual patient as their disease evolves and progresses in space and time. There is a recognised diversity in clinical behaviour. Subsets of patients with high-risk phenotypes, including early progressors and those who experience transformation to a high-grade lymphoma, remain priority areas of unmet need given the inferior outcomes,3,4 although the molecular drivers behind such clinical behaviours remain incompletely understood.

The Mutation Landscape of Follicular Lymphoma and Chronic Lymphocytic Leukemia Reveals a Distinctive Frequency and Composition of Aid-Related Mutation Signatures

Introduction: Somatic mutations associated with distinctive types of cancer can be globally classified by the trinucleotide context of single nucleotide variants into mutational signatures. Distinct signatures can be attributed to underlying causative mechanisms such as UV irradiation or smoking. B-cell lymphomas have only been analyzed globally and carry a signature characterized by APOBEC activity (Alexandrov, 2014).The two most common indolent B-cell neoplasms, chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), are characterized by distinctive pathogenic processes, but mutagenesis caused by the APOBEC enzyme activation-induced cytosine deaminase (AID) may be shared by both malignancies. AID deaminates deoxycytidine to deoxyuridine, which is processed by DNA repair enzymes into a larger spectrum of point mutations and also DNA double-strand breaks. AID activity and subsequent DNA repair create two different mutation signatures: the canonical signature characterized by C>T/G transitions at RCY motifs and the non-canonical defined by A>C transversions at WAN motifs. More recently a third AID signature characterized by C>T transitions at RCG motifs has been associated with AID-mediated CpG-methylation dependent mutagenesis.We here investigate the hypothesis that AID mutational signatures contribute to the pathogenesis of FL, a lymphoma characterized by constitutional expression of AID, but also of CLL since high AID expression levels may account for a more aggressive disease, and a subset of CLL B-cells with AID expression display dissociation between class switch recombination and somatic hypermutation.Methods: We analyzed the mutational landscape in whole exome sequencing data from 31 FL and 24 CLL cases. Somatic variants were called by comparison of tumor and germline DNA with an in-house developed pipeline. The relative contribution of mutations at canonical and non-canonical AID hotspot motifs was computed by direct quantification of variants occurring in known AID motifs. Student's t test was used for comparison between CLL and FL.Mutational signatures were defined according to the 96-base substitution model (Alexandrov, 2013) by an unsupervised machine learning approach, with a local implementation of the SomaticSignatures R package.Results: The frequency of C>T/G transitions at canonical AID hotspot was more prevalent in CLL (9.0%; 95% CI: 8.1-10.0%) than in FL (6.6%, 95% CI: 5.9-7.3%)(p<0.001). Likewise, A>C transversions at non-canonical AID sites were significantly more frequent in CLL (1.9%; 95% CI: 1.6-2.2%) as compared to FL (0.5%; 95% CI: 0.4-0.6%)(p<0.0001).In the unsupervised analysis of FL and CLL cases, 97.7% of the mutation spectrum variance was explained by only 5 signatures (Figure 1). In FL, the mutational landscape was dominated by a signature (S2) characterized by C>A transversions in the trinucleotide KCH context (40%, 95% CI: 33-36%). The second most frequent signature (S3; 22%, 95% CI: 15-30%) was characterized by C>T transitions in the context of a hybrid between the canonical AID and the CpG hotspot motifs (RCY/RCG).In contrast, the mutational landscape of CLL was dominated by a signature (S5; 42%, 95% CI: 34-49%) characterized by C>T transitions in the RCY motif and A>C transversions in the WAN context, consistent with an overlapping between the canonical and the non-canonical AID hotspots (Kasar, 2016).Conclusions: The mutational landscape of both CLL and FL seems to be strongly shaped by AID activity. In FL, AID-induced mutations are mainly restricted to canonical AID hotspots and CpG-methylation dependent mutagenesis sites. In strong contrast, both canonical and non-canonical AID motifs contributed to the mutational landscape of CLL. In addition, FL carries a high prevalence of C>A substitutions that are associated with exposure to polycyclic aromatic hydrocarbons that produce bulky DNA adducts followed by the nucleotide excision repair process. These compounds are present in several industrial processes (food products, beverages, tobacco) that have been suggested as occupational risk factors for follicular lymphomas (Mester, 2009).Overall, our data suggest a much stronger role of AID in CLL pathogenesis than hitherto appreciated. The differential contribution of canonical versus non-canonical AID activity points to different regulation of AID in CLL compared to FL and warrants further investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Microsatellite instability (MSI, EMAST) in the pathogenesis of follicular lymphoma

Background. Genetic instability, an important phenomenon involved in oncogenic transformation and tumor progression, is associated with the insufficiency of the multicomponent DNA repair complex, in particular, the nucleotide mismatch repair (MMR) system. The MMR defect manifests itself as abnormalities in DNA microsatellite repeats, or microsatellite instability (MSI). In the studies of colorectal cancer, the role of MSI in prognostication of the disease, and defining the choice of specific therapy with immune checkpoint inhibitors has been proven.However, in lymphatic system tumors, the significance of this phenomenon is poorly understood. Determination of genetic instability in the onset of follicular lymphoma, a disease characterized by a heterogeneous course, may have prognostic value.Objective: to determine the genetic instability at the onset of follicular lymphoma.Materials and methods. Here we report an analysis of 24 microsatellite repeats and amelogenin loci in tumor cells of 46 follicular lymphoma patients.Results. In the studied cohort, lesions in microsatellite repeats were presented by MSI in 9 cases (19.6 %) and the loss of heterozygosity (LOH) in 19 cases (41.3 %). Most frequent lesions were found for the SE33 marker located at the q14 locus of chromosome 6. A significant association was shown between MSI and the double-hit follicular lymphoma group with rearrangements of the MYC and BCL2/BCL6 genes.Conclusion. Thus, our data indicate that the MSI phenomenon might be involved in the pathogenesis of the lymphatic tumors and particularly follicular lymphoma. However further studies on the expanded cohorts of patients are required to define the possible prognostic value of MSI in lymphatic tumors.

Molecular insights into the pathogenesis of follicular lymphoma

: Follicular lymphoma (FL) represents a group of B-cell neoplasms derived from germinal center (GC) B cells. A better understanding of the pathogenic mechanisms of FL is important for developing innovative therapies. The most common form of FL is primarily nodal and associated with the t(14;18). Recent advances obtained via genomic profiling have provided unprecedented insights into the pathogenesis of FL. Conventional FL evolves through multiple independent or convergent genetic pathways. The classical pathogenesis of t(14;18)-positive FL is a multi-stage and multi-hit process escalating along accumulation of genetic and epigenetic alterations, which starts from FL-like B cells, through premalignant lesions, into full-blown malignancy. Early precursor lesions have been recognized in the form of FL-like B cells in normal peripheral blood, and both in-situ follicular neoplasia (ISFN) and duodenal-type FL. In comparison, t(14;18)-negative FL is much more heterogeneous at the molecular level, and the underlying mechanisms are less well understood. Some variants of FL, while lacking upregulation of BCL2, share a common mutational profile with conventional FL, including mutations in epigenetic modifiers. These cases also show some clinical overlap with BCL2-positive FL, including mainly nodal involvement. Other forms of FL show more profound differences, both clinically and biologically. These emerge more clearly as separate entities and include FL, grade 3B, testicular FL (TFL), pediatric-type FL (PTFL), and primary cutaneous follicle center lymphoma (PCFCL). Mutations in epigenetic regulators and 1p36/TNFRSF14 abnormalities are highly recurrent and are seen across different subtypes of FL. Genetic profiling has offered important new insights, and will continue to impact the diagnostic approach, with changes in future classification schemes.