Pathogenesis Of Follicular Lymphoma Research Articles

Follicular B Lymphomas Generate Regulatory T Cells via the ICOS/ICOSL Pathway and Are Susceptible to Treatment by Anti-ICOS/ICOSL Therapy

The prognosis of follicular lymphoma (FL) patients is suspected to be influenced by tumor-infiltrating regulatory T cells (Treg). The mechanism of Treg enrichment in FL and their impact on malignant FL B cells remains to be elucidated. We analyzed 46 fresh lymph node biopsy samples, including FL (n = 20), diffuse large B-cell lymphoma (n = 10), classical Hodgkin lymphoma (n = 9), and reactive lymphadenitis (n = 7). Using multicolor flow cytometry and cell sorting, we observed an accumulation of CD25(high)CD127(low/neg) Tregs in FL tissues. These Tregs comprised activated ICOS(+) Tregs that were able to suppress not only conventional T cells, but also FL B cells. These FL B cells were able to express ICOSL in vitro and to generate CD25(high)FoxP3(high) Tregs expressing ICOS. Treg generation was associated with ICOS/ICOSL engagement and was abrogated by antagonist anti-ICOS and anti-ICOSL antibodies. Interactions between Tregs and FL B cells resulted in ICOSL downregulation on FL B cells. Our results highlight a key role for Tregs in FL pathogenesis and suggest that targeting the ICOS/ICOSL pathway may be a promising immunotherapy for FL treatment. Cancer Res; 76(16); 4648-60. ©2016 AACR.

MicroRNA expression profiling of Chinese follicular lymphoma by microarray: A preliminary study

BackgroundMicroRNAs (miRNAs) have been widely regarded as crucial regulators in various biological processes involved in carcinogenesis. However, the comprehensive miRNA profiles of Chinese follicular lymphoma (FL) remains completely unknown. MethodsThe Exiqon miRCURY LNA™ microRNA Array (v.18.0) was used to detect the miRNA expression profiles of three Chinese FL samples, and compared to three reactive lymphatic nodes (RLN). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to confirm the selected miRNAs in different series. Three databases (miRAnda, miRBase and TargetScan) were used to predict the putative target genes. Bioinformatic analysis (gene ontology analysis and pathway analysis) was performed for further evaluation. ResultsThe microarray assay demonstrated that 1643 miRNAs were expressed; in which 103 miRNAs were upregulated and 68 miRNAs were downregulated, according to P-value (<0.05) and fold change (FC>2-fold). Furthermore, qRT-PCR was used to confirm that miR-17-5p, miR-20a-5p and miR-19a-3p were upregulated, and miR-3615 was downregulated (P<0.05). Bioinformatic analysis (gene ontology analysis and pathway analysis) was used for further evaluation. Pathway analysis indicated that 25 pathways corresponded to differentially expressed miRNAs (P-value cut-off is 0.05). Furthermore, miR-17-5p, miR-20a-5p and miR-19a-3p were validated by qRT-PCR in an independent series including five FL3a and five RLN cases. Data analysis revealed that the changing trend of miR-19a-3p and miR-17-5p expression in the independent series was basically identical with that of the microarray data. ConclusionsOur results are the first to reveal the miRNA expression profiling of Chinese FL and three upregulated miRNAs. Furthermore, the expression of miR-19a-3p and miR-17-5p were found to be significantly upregulated in FL3a. Further study needs to be urgently performed to reveal its potential role in the pathogenesis of FL in the near future.

Integrated immunohistochemical and DNA copy number profiling analysis provides insight into the molecular pathogenesis ofcanine follicular lymphoma.

Follicular lymphomas (FLs) typically exhibit a chromosome translocation that induces constitutive expression of the anti-apoptotic bcl2 protein and accumulation of additional molecular defects. This rearrangement offers a promising therapeutic target, but its nature as a fundamental driver of FL pathogenesis remains unclear as 15% of cases lack the translocation. We performed an integrated immunohistochemical and genomic investigation of 10 naturally occurring FL cases from domestic dogs, showing that, as with human tumours, they exhibit marked heterogeneity in the frequency and intensity of bcl2 protein expression. Genomic copy number aberrations were infrequent and broadly consistent with those of other canine B-cell lymphoma subtypes. None of the canine FL specimens exhibited a rearrangement consistent with the hallmark translocation of human FL, despite their remarkable histomorphologic similarity. Parallel exploration of canine and human cases may reveal alternative tumour-initiating mechanisms other than BCL2 disruption, yielding a more complete definition of the molecular pathogenesis of FL.

Analysis of 54 Follicular Lymphomas By Whole Exome Sequencing Identifies Multiple Novel Recurrently Mutated Pathways

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. FL carries multiple recurrently mutated genes that are under active investigation. However, due to the relatively small number of published sequenced cases, knowledge regarding the coding genome in FL is still evolving.Methods: To further our understanding of the genetic basis of FL, we used solution exon capture of sheared and processed genomic DNA isolated from highly purified light chain restricted B-cells and paired CD3+ T-cells from 54 FL cases for paired-end massively parallel sequencing (WES). Data were subsequently analyzed using bioinformatics pipelines including the variant callers MuTect v.1.1.4, Strelka v.1.0.13, and VarScan2 v.2.3.7. Candidate somatically acquired gene mutations with variant allele frequencies (VAFs) >0.15 were confirmed using Sanger sequencing. Selected mutations were validated in an expansion cohort of 120 FL.Results: We identified heterozygous missense mutations in the mTOR regulator RRAGC in 10% of FL. The RRAGC mutations targeted multiple hotspot residues (amino acid 115, 118 and 119). RRAGC forms heterodimers with either RRAGA or RRAGB that under conditions of amino acid sufficiency facilitate recruitment of mTOR through the raptor subunit to lysosomal membranes. At the lysosomal surface, multiple protein complexes, each containing various proteins regulate mTOR activation through RHEB.To gain insights into the functional consequences of RRAGC mutations, we performed 3-dimensional modeling of FL-associated RRAGC mutations and located the mutations into relatively close proximity to the RRAGC GTP/GDP binding site. Energy calculations did not identify strong effects of mutated amino acid residues on the binding of GTP/GDP to RRAGC.We performed studies of the effects of RRAGC mutants on mTOR activity as measured through S6-kinase phosphorylation. In transient transfection systems (293T and HELA) achieving expression slightly above endogenous RRAGC levels, performed under conditions of leucine starvation or sufficiency, we did not identify differences in baseline mTOR activation. In stably transfected 293T cell lines (expressing RRAGB and RRAGC proteins above endogenous levels), that were starved for leucine for 1 hour, we detected modestly elevated p-S6K levels in RRAGC mutant versus wild type transfectants, suggesting a mild intrinsic activation phenotype of RRAGC mutations. Experiments in lentivirally-transfected lymphoma cell lines, including RRAGC binding studies to raptor and folliculin (a RRAGC regulator) are in progress and will be updated at the meeting. Curiously, we did not identify mutations in the other three small GTP binding proteins that are part of the same amino acid sensing pathway (RRAGA, RRAGB or RRAGD), potentially pointing to a unique advantage conferred by RRAGC mutants on FL B cells.We identified additional mutations (combined ~15%) in other mTOR components linked to lysosomal amino acid sensing, including recurrent mutations in the v-ATPase subunit ATP6V1B2 and the accessory subunit ATP6VAP1. The mutations in RRAGC and v-ATPase together highlight a previously unidentified role of the amino acid sensing pathway that regulates mTOR in FL pathogenesis.We have discovered a high frequency of mutations (40%) in the surrogate light chain gene IGLL5 in FL, a critical component of the pre-B-cell receptor. Mutations sharply cluster in the N-terminal 70 amino acid of IGLL5, a region known as the non-Ig domain of IGLL5. The non-Ig domain of IGLL5 has been implicated in influencing pre-B-cell receptor signaling and receptor surface expression as well as interaction with extracellular ligands. The mutational data suggest an unexpected role of IGLL5 in the pathogenesis of FL and work is in progress studying IGLL5 expression in primary FL samples.Conclusion: This large WES study of 54 FL identifies novel recurrently mutated genes and pathways in FL, including frequent mutations in genes involved in amino acid signaling to mTOR (RRAGC and v-ATPase) as well as pre-B-cell receptor signaling (the surrogate light chain gene IGLL5) and multiple other novel recurrently mutated genes that will be updated at the meeting. These data substantially broaden our understanding of the genetic basis of FL and provide clues to therapeutically targeting specific pathways in FL. DisclosuresMalek:Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership; Janssen Pharmaceuticals: Research Funding.

Follicular Lymphoma B Cells Generate Functional Regulatory T Cells Via ICOS/ICOSL Pathway and Are Inhibited By Intratumoral Tregs

The prognosis of follicular lymphoma (FL) patients is suspected to be influenced by tumor infiltrating regulatory T cells (Tregs). The mechanism of Tregs enrichment in FL and their impact on malignant FL B cells remains to be elucidated. We analyzed 46 fresh lymph node biopsy samples including FL (n = 20), diffuse large B cell lymphoma (DLBCL; n = 10), classical Hodgkin lymphoma (cHL; n = 9) and reactive lymphadenitis (rL; n = 7). Using multicolor flow cytometry and cell sorting, we observed an accumulation of CD25high CD127low/- Tregs in FL tissues. These Tregs comprised activated ICOS+ Tregs which were able to suppress not only conventional T cells, but also FL B cells. These FL B cells were able to express ICOSL in vitro and to generate CD25high Foxp3high Tregs expressing ICOS. Tregs generation was associated with ICOS/ICOSL engagement and was abrogated by antagonist anti-ICOS and anti-ICOSL antibodies. Interaction between Tregs and FL B cells resulted in ICOSL downregulation on FL B cells. These data highlight the role of Tregs in FL pathogenesis and suggest that targeting the ICOS/ICOSL pathway may be a promising immunotherapy in FL DisclosuresNo relevant conflicts of interest to declare.

Lectins from opportunistic bacteria interact with acquired variable-region glycans of surface immunoglobulin in follicular lymphoma

AbstractB-cell antigen receptor (BCR) expression is a key feature of most B-cell lymphomas, but the mechanisms of BCR signal induction and the involvement of autoantigen recognition remain unclear. In follicular lymphoma (FL) B cells, BCR expression is retained despite a chromosomal translocation that links the antiapoptotic gene BCL2 to the regulatory elements of immunoglobulin genes, thereby disrupting 1 heavy-chain allele. A remarkable feature of FL-BCRs is the acquisition of potential N-glycosylation sites during somatic hypermutation. The introduced glycans carry mannose termini, which create potential novel binding sites for mannose-specific lectins. Here, we investigated the effect of N-linked variable-region glycosylation for BCR interaction with cognate antigen and with lectins of different origins. N-glycans were found to severely impair BCR specificity and affinity to the initial cognate antigen. In addition, we found that lectins from Pseudomonas aeruginosa and Burkholderia cenocepacia bind and stimulate FL cells. Human exposure to these bacteria can occur by contact with soil and water. In addition, they represent opportunistic pathogens in susceptible hosts. Understanding the role of bacterial lectins might elucidate the pathogenesis of FL and establish novel therapeutic approaches.

Influence of reproductive history and exogenous hormone use on prevalence and frequency of circulating t(14;18)-positive cells in a population-based cross-sectional study.

PurposeThe t(14;18) translocation might represent an intermediate step in the pathogenesis of follicular lymphoma (FL), one of the most common subtypes of non-Hodgkin lymphoma. Circulating t(14;18)-positive cells can also be detected in 30–60 % of healthy individuals at low frequencies. Some studies found a negative association between reproductive factors or use of menopausal hormone therapy (MHT) with FL. The objective of this study was to evaluate whether there is an association between number of frequencies, oral contraceptive (OC) use, menopausal status and MHT, and t(14;18) prevalence and frequency in a representative population analysis based on an epidemiologic study in the northeastern part of Germany.MethodsThe analysis is based on results of buffy coat samples from 1,981 women of the Study of Health in Pomerania (SHIP-0) and data obtained in standardized face-to-face interviews. For prevalence, odds ratios (OR) and 95 % confidence intervals (CI) were calculated using unconditional logistic regression. Frequency data were analyzed using negative binomial regression. The multivariable models included age, number of pregnancies, menopausal status (premenopausal, natural, medical/surgical menopause), OC use and MHT as a measure for exogenous hormone exposure use.ResultsWe found no association between reproductive history and combined exogenous hormone use on the prevalence of circulating t(14;18)-positive cells. Modeling MHT and OC use separately in a sensitivity analysis, the MHT parameter yielded statistical significance [OR 1.37 (95 % CI 1.04;1.81)]. t(14;18) frequency was associated with use of OC [incidence rate ratio (IRR) for ever use 3.18 (95 % CI 1.54;6.54)], current use [IRR 3.86 (1.56;9.54)], >10 years use [IRR 3.93 (1.67;9.23)] and MHT [restricted to postmenopausal women; IRR 2.63 (95 % CI 1.01;6.85)] in bivariate age-adjusted analyses. In the multivariable model, medical/surgical menopause [IRR 2.46 (1.11;5.44)] and the category ever use of OC and MHT were statistically significant [IRR 2.41 (1.09;5.33)].ConclusionsExogenous hormone use might be a risk factor for t(14;18) frequency rather than for t(14;18) prevalence. Further research on healthy individuals carrying a t(14;18) translocation and possible risk factors for malignant lymphoma is necessary to determine the additional molecular or immunological events that have to occur to develop FL.Electronic supplementary materialThe online version of this article (doi:10.1007/s10552-015-0525-4) contains supplementary material, which is available to authorized users.

Multiparameter Microscopy Analysis of the Follicular Lymphoma Microenvironment and Normal Germinal Centers: In Vivo evidence That Follicular Helper T Cells Form Synapses with Neoplastic B Cells and Are Associated with Proliferation and Expression of Activation Induced Cytidine Deaminase

The tumor microenvironment plays a central role in the pathogenesis of follicular lymphoma (FL) and has been shown to influence prognosis. The biological basis for this and the contribution of individual cell types however, remain unclear. In this study we compared the cellular content and structure of neoplastic follicles in FL with their normal counterparts in reactive lymph nodes (LNs). We specifically focused on follicular helper T cells (TFH) which, in normal germinal centers (GCs), form immune synapses with antigen responsive B cells triggering B cell proliferation and expression of activation induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class switch recombination. This is of relevance because off-target AID activity is thought to play a role in generating the mutations that characterize progressive FL.A limitation of previous studies of the FL microenvironment is the use of either single parameter immunohistochemistry which fails to accurately define the complex populations of cells involved, or flow cytometry on disaggregated cells which results in the loss of architectural information. In this study we used multiparameter confocal immunofluorescent (IF) microscopy to investigate in vivo the phenotype, distribution and interaction of CD4+ T cells in FL and to determine to what extent these are similar to normal GCs.Confocal IF microscopy was performed on multiple sections of formalin fixed paraffin embedded LN biopsy specimens from 20 patients with untreated FL, comparison was made with reactive LNs (n=5) and chronic lymphocytic leukemia (CLL) LN biopsies (n=5). Each section was stained with a combination of up to 4 simultaneously applied primary antibodies against CD3, CD4, CD20, PD1, ICOS, BCL6, AID, and Ki67, and fluorescently labelled secondary antibodies. Microscopy was performed using a Nikon TiE fluorescent microscope equipped with A1R Si Confocal imaging system; images were analyzed using NIS software.Results show that CD4+ T cells in FL are mainly located in the inter-follicular regions but they were also identified within the follicles in all cases. Combination staining with anti-CD4, PD1, and ICOS revealed that 23% (95%CI 18-27) of CD4+ T cells within follicles co-express PD1 and ICOS consistent with a TFH phenotype which is significantly higher than in inter-follicular areas where only 5% (95% CI 3-7) of CD4+ cells had this phenotype (p<0.001). PD1+ ICOS+ T cells were positive for the transcription factor BCL6, further confirming the TFH phenotype. There was no significant difference in the proportion of CD4+ cells that were TFH in FL follicles and reactive LN GCs. In CLL cases, 54% of CD4+ cells expressed PD1 but only 9% co-expressed PD1 and ICOS, significantly lower than either FL follicles or GCs (p<0.001). Automated analysis of 3D z-stacks demonstrated a very close spatial relationship between proliferating tumor cells and TFH in FL with a mean of 42% (95%CI 35-48) Ki67+ tumor cells in direct contact with TFH cells. No association was seen between the extent of co-localization and histological grade. A similar pattern of co-localization of TFH cells next to proliferating B cells was also identified in the light zones of reactive GCs. Of note, we also identified features of synapse formation between TFH cells and proliferating tumor cells; TFH cells demonstrated projections that encompass the tumor cell with distortion of the T cell nucleus and increased CD4 and PD1 expression at sites of cell contact (Figure 1). These findings were similarly present in reactive GCs. Finally, AID was expressed in proliferating GC B cells and in proliferating tumor cells in FL. AID expressing cells were found to be in close contact with PD1+ T cells in both GCs and FL.Our findings show many parallels between the follicles of FL and normal GCs. In particular the proportion of CD4+ T cells with a TFH phenotype and their localization in direct contact with proliferating AID+ B cells were very similar. Of note, features of immune synapses were observed in both GCs and FL. Taken together, the data suggest that TFH cells have an important role in the pathogenesis of FL just as they are vital in the normal GC reaction. Interruption of this interaction is a potential therapeutic target. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

FAS Mutations Accelerate Lymphoma Growth and Induce Therapeutic Resistance

▪ BACKGROUNDRituximab-based chemotherapy is effective in inducing remissions in ~85% patients with untreated follicular lymphoma (FL). Primary treatment failure or an early relapse after first-line therapy is associated with a very poor prognosis. Recent sequencing efforts have been successful at identifying recurrent genetic events that contribute to the pathogenesis of FL, but the clinical significance of most of these events, in particular those contributing to therapeutic resistance, remain unknown. We identified a mutation in FAS (Y232*) in a patient with primary-refractory FL. FAS, a key death receptor in the extrinsic apoptotic pathway, plays a fundamental role in immune homeostasis by initiating apoptosis in lymphocytes once activated by FAS ligand (FASL) from neighboring cells. Herein, we show that mutations in FAS contribute to therapeutic resistance in FL. METHODSWe determined the incidence and clinical significance of FAS mutations in an extended cohort of 214 clinically-annotated FL biopsies. We determined the impact of the recurrent FAS(Y232*) mutation on FAS-mediated and chemotherapy-induced apoptosis in lymphoma cell lines. We measured the change in FAS and FASL expression in primary human T and B lymphocytes after exposure to chemotherapy. Finally, we cloned the murine equivalent of FAS(Y232*), Fas(Y224*), in Eu-Myc lymphoma cells and determined its effect on lymphoma growth and response to chemotherapy in immunocompetent C57BL/6 mice (n=36). RESULTSFAS mutations were identified in 6% of FL patients. Coding FAS mutations were associated with a trend towards an earlier median time to progression (1 y versus 2.8 y, p=0.08) and an increased risk of histological transformation (p=0.036). The recurrent FAS(Y232*) mutation inhibited FAS-mediated apoptosis in cell lines but, unexpectedly, did not inhibit chemotherapy-mediated apoptosis. We hypothesized that in patients, chemotherapy induced a FAS-mediated immune response that was not modeled in vitro. Supporting this concept, we observed an increase in FASL and FAS expression on normal T and B lymphocytes, respectively, after exposure to etoposide. We injected three groups of mice with Eu-Myc lymphoma cells that differed only in their Fas genotype (Fas WT, Fas(Y224*) and an empty vector control) and monitored lymph node volumes before and after therapy. Fas(Y224*) dramatically accelerated lymphoma growth. Lymph node volumes exceeded those measured in Fas wild-type and control mice at all time points beyond the day of injection. The average maximal lymph node volume for the Fas(Y224*) group was 59.9 mm3 compared to 18.9 mm3 and 34.5 mm3 for the Fas WT and control groups, respectively (p < 0.001). Fas mutant lymphomas had an inferior response to doxorubicin and none of the mice in this group achieved a complete remission. Remarkably, the opposite phenotype was observed in the Fas WT group, where the addition of Fas WT inhibited lymphoma growth and induced earlier remissions. CONCLUSIONMutations in FAS can be clinically important in patients with FL by promoting lymphoma growth and inducing therapeutic resistance. The full malignant phenotype of FAS mutant lymphomas could only be elicited in vivo, and not in vitro, suggesting that a FAS-mediated immune response controls lymphoma growth and actively participates in chemotherapy-induced cell death. DisclosuresConnors:Seattle Genetics, Inc.: Research Funding; Roche: Research Funding.

Indolent lymphoma: follicular lymphoma and the microenvironment-insights from gene expression profiling.

As shown with gene expression profiling (GEP), the development and progression of follicular lymphoma (FL) involves complex interactions between neoplastic B cells and the surrounding microenvironment. GEP further reveals that the tumor microenvironment may predict survival in patients with FL and influence the response to therapy and the risk of transformation. Here, we briefly review GEP technology and summarize the role of the tumor microenvironment in FL diagnosis, prognosis, and transformation. Genes expressed by infiltrating T cells and macrophages appear to be the most important predictors of survival, clinical behavior, and outcome. These findings provide a basis for future studies into the pathogenesis and pathophysiology of FL and may ultimately provide guidance in the choice of therapy and the identification of potential therapeutic targets.

Abstract 5072: Meta-analysis of genome-wide association studies identifies novel susceptibility loci for follicular lymphoma

Abstract Background. Follicular lymphoma (FL) is an indolent B-cell malignancy with a variable clinical course that may transform to more aggressive forms of lymphoma. Previous modestly powered GWAS have reported multiple FL susceptibility loci in the HLA class I and II regions at 6p21.32-33, but novel non-HLA susceptibility loci and the genetic architecture of FL associations in the HLA region remain to be elucidated. Methods. We conducted the largest GWAS of FL to date consisting of 2,142 cases and 6,221 controls of European ancestry from 22 studies scanned on the Illumina OmniExpress. After imputation of common SNPs using IMPUTE2 and 1,000 Genomes Project v3 data, we conducted a meta-analysis of these data with two previous FL GWAS (n=586 cases, 1,537 controls) and replicated top hits in an additional 629 cases and 4,283 controls. Expression quantitative trait loci (eQTL) analyses were performed to evaluate the effects of associated variants on gene expression. Classical HLA allele imputations and stepwise regression analyses are underway to further characterize the HLA associations. Results. In the joint meta-analysis, the strongest association with FL risk was observed in the HLA region, where a large number of SNPs showed genome-wide significance (P&amp;lt;5x10-8 to P= 1.84x10-84), particularly in HLA class II at 6p21.32 where several HLA eQTLs were identified (P &amp;lt; 0.05). Outside HLA, we identified four novel loci associated with FL at 11q23.3 (CXCR5, rs4938573; P=3.14x10-15), 11q24.3 (ETS1, rs4937362; P=3.33x10-9), 3q13.33 (CD86, rs2681416; P=9.79x10-11) and 3q28 (LPP, rs6444305, P=2.55x10-8). These novel susceptibility loci were located in four biologically relevant genes involved in immune regulation: CXCR5 and its ligand, CXCL13, are involved in guiding B-cells into the B-cell zones of secondary lymphoid organs as well as T-cell migration. ETS1 is expressed in lymphoid cells and regulates immune cell function including differentiation, survival, and proliferation. CD86 and CD80 are classic members of the B7 costimulatory pathway that is important in maintaining immune function, suppression of autoimmunity and antitumor surveillance. LPP encodes a LIM domain-containing protein of the zyxin family and participates in cell adhesion, cell migration, proliferation and transcription dynamics. Conclusions. This large GWAS provides further support for the important role of common genetic variation in non-HLA genes and further evidence of the key role that HLA immune-regulatory genes play in the pathogenesis of FL. Citation Format: Christine F. Skibola, Sonja I. Berndt, James R. Cerhan, Zhaoming Wang, Joseph Vijai, Lucia Conde, Paul de Bakker, Sophia S. Wang, Claire M. Vajdic, Brenda M. Birmann, Susan L. Slager, James McKay, Paige M. Bracci, Alexandra Nieters, Qing Lan, Angela R. Brooks-Wilson, Martha S. Linet, Demetrius Albanes, John J. Spinelli, Roel C.H. Vermeulen, Mark P. Purdue, Meredith Yaeger, Lauren R. Teras, Silvia de Sanjose, Alain Monnereau, Simon Crouch, Jia Nee Foo, Henrik Hjalgrim, Gianluca Severi, Brian K. Link, Kimberly A. Bertrand, Yawei Zhang, Karin E. Smedby, Stephen J. Chanock, Nathaniel Rothman, NHL GWAS Consortium. Meta-analysis of genome-wide association studies identifies novel susceptibility loci for follicular lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5072. doi:10.1158/1538-7445.AM2014-5072

Constitutive AKT activation in follicular lymphoma

BackgroundThe phosphoinositide 3- kinase (PI3K) pathway is involved in the growth of various human cancers, including lymphoid malignancies. However its role in the pathogenesis of follicular lymphoma (FL) has not been yet described.MethodsTo clarify this point, biopsy tissue samples from 38 human FL cases were investigated for PIK3CA somatic mutations in exon 9 and 20 using direct sequencing. The same samples were analyzed using western blotting and immunohistochemistry to detect expression of AKT, phosphorylated AKT (pAKT), and PTEN proteins. Two cases of benign lymphadenitis were used as controls.ResultsAKT expression was present in all FL and lymphadenitis cases. 14/38 (37%) FL and 2/2 lymphadenitis cases expressed pAKT. 9/38 (24%) FL samples showed high level of pAKT, whereas 5/38 (13%) FL cases and 2/2 benign lymphadenitis samples expressed low level of pAKT. PTEN expression was observed in 30/38 (79%) FL and 2/2 benign lymphadenitis cases, whereas 8/38 (21%) FL cases showed loss of PTEN expression. 3 cases with positive pAKT did not express PTEN. PIK3CA mutations were not detected in any sample.ConclusionsThese data suggest that the PI3K/AKT signaling pathway could be activated in a subset of FL cases, due to either AKT phosphorylation or PTEN downregulation, in the absence of PIK3CA mutations.

Mutations in linker histone genes HIST1H1 B, C, D, and E; OCT2 (POU2F2); IRF8; and ARID1A underlying the pathogenesis of follicular lymphoma

AbstractFollicular lymphoma (FL) constitutes the second most common non-Hodgkin lymphoma in the western world. FL carries characteristic recurrent structural genomic aberrations. However, information regarding the coding genome in FL is still evolving. Here, we describe the results of massively parallel exome sequencing and single nucleotide polymorphism 6.0 array genomic profiling of 11 highly purified FL cases, and 1 transformed FL case and the validation of selected mutations in 102 FL cases. We report the identification of 15 novel recurrently mutated genes in FL. These include frequent mutations in the linker histone genes HIST1H1 B-E (27%) and mutations in OCT2 (also known as POU2F2; 8%), IRF8 (6%), and ARID1A (11%). A subset of the mutations in HIST1H1 B-E affected binding to DNMT3B, and mutations in HIST1H1 B-E and in EZH2 or ARID1A were largely mutually exclusive, implicating HIST1H1 B-E in epigenetic deregulation in FL. Mutations in OCT2 (POU2F2) affected its transcriptional and functional properties as measured through luciferase assays, the biological analysis of stably transduced cell lines, and global expression profiling. Finally, multiple novel mutated genes located within regions of acquired uniparental disomy in FL are identified. In aggregate, these data substantially broaden our understanding of the genomic pathogenesis of FL.

Molecular Pathogenesis of Follicular Lymphoma

t(14;18) translocation has been recognized as a genetic hallmark of follicular lymphoma (FL), but it is now known that additional genetic aberrations are required for the development of FL. With recent advances in the technology for DNA analysis, recurrent gene aberrations such as TNFRSF14, EPHA7, EZH2, CREBBP, EP300, MLL2 and MEF2B have been identified. A few t(14;18)-positive B cells can be detected in healthy individuals, and these B cells are reported to have their own biological features that are closely associated with the pathogenesis of FL. On the other hand, FL is characterized by a unique microenvironment. Further understanding of the pathogenesis of FL is expected to contribute to the development of novel treatment approaches for this disease.

Investigating The Role Of MLL2 (Mll4) In B Cell Development

BackgroundThe myeloid/lymphoid or mixed-lineage leukaemia 2 (MLL2) histone methyltransferase (referred to as Mll4 in mice) forms part of a large multiprotein complex, which catalyses the methylation of lysine 4 on histone H3 (H3K4). High levels of this histone modification are detected at promoter regions of actively transcribed genes. Loss-of-function mutations in MLL2 have been identified in 80-90% of follicular lymphoma (FL) cases. These mutations are distributed throughout the coding region and lead to the loss of the C-terminal catalytic SET domain and reduction in H3K4 methylation. We generated a Mll4 knockout (Mll4-/-) mouse model to elucidate the effects of Mll4 loss on B cell development and understand how mutations in this gene contribute to FL pathogenesis. Results and DiscussionCre:ERT2-mediated recombination was used to induce the deletion of exons 2–4 of Mll4 in adult mice. CD19+ B lymphocytes were purified from the spleens of wild-type (Mll4WT) and Mll4-/- mice and confirmed the loss of Mll4 mRNA and protein by quantitative RT-PCR (qRT-PCR) and immunoblotting, respectively. As expected, B cells lacking Mll4 (Mll4ΔB/ΔB) exhibited a global reduction in H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3), compared with normal B cells. Gene expression profiling (GEP) using the GeneChip® Mouse Genome 430 2.0 Array (n=5 Mll4WT + 5 Mll4-/- mice) was carried out to determine the transcriptional changes upon loss of Mll4. We identified >200 genes differentially expressed (>2-fold) between Mll4WT and Mll4ΔB/ΔB CD19+ B cells. The top 40 candidate genes (p<0.05) were verified in an independent series of experiments using qRT-PCR. We noted a significant decrease in the expression of the transcription factor lymphoid enhancer-binding factor 1 (Lef1) and the histone acetyltransferase nuclear receptor coactivator 3 (Ncoa3). The downregulation of these genes is consistent with published data, as the promoters of both are marked by H3K4me3 in normal mouse CD19+ B cells. Lef1 is a key regulator of lymphoid differentiation, while Ncoa3 depletion has been shown to induce B-cell lymphoma in mice as a result of constitutive NF-κB activation. A significant number of genes (p<0.001) involved in cell cycle and immune response were upregulated in Mll4ΔB/ΔB CD19+ B cells. Furthermore, loss of Mll4 led to an elevated expression (2.4-fold) of activation-induced cytidine deaminase (Aicda), an enzyme required for germinal centre-derived lymphomagenesis. Immunophenotyping was used to examine the role of Mll4 at different stages of B cell development. We detected a significant reduction in the number of pre-B cells (B220+CD43–IgM–) in the bone marrow of Mll4-/- mice (n=12), compared with their littermate controls (n=10; p<0.01), although this did not affect the number of peripheral mature splenic B cells (B220+IgM+). Mll4 loss had an adverse effect on immune response, with CD19+ B cells failing to induce expression of the MHC-II, CD86, CD40 and CD69 activation markers upon in vitro stimulation with lipopolysaccharide and interleukin 4. ConclusionOur findings provide the first insight into the potential mechanistic link between MLL2 loss and the onset of FL using a mouse model. Mll4-/- mice do not develop any lymphoproliferative disorders, but show defects in B cell development and an impaired immune response. Furthermore, loss of Mll4 leads to the global depletion of H3K4 methylation in mouse B lymphocytes, thus affecting the expression of Lef1, Ncoa3 and Aicda. Although these MLL2/Mll4 target genes have defined roles in B cell biology, their contribution to the pathogenesis of FL will depend on when MLL2 mutations arise during FL development. Disclosures:No relevant conflicts of interest to declare.

Early lesions of follicular lymphoma: a genetic perspective

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

Coding Variants at Hexa-allelic Amino Acid 13 of HLA-DRB1 Explain Independent SNP Associations with Follicular Lymphoma Risk

Non-Hodgkin lymphoma represents a diverse group of blood malignancies, of which follicular lymphoma (FL) is a common subtype. Previous genome-wide association studies (GWASs) have identified in the human leukocyte antigen (HLA) class II region multiple independent SNPs that are significantly associated with FL risk. To dissect these signals and determine whether coding variants in HLA genes are responsible for the associations, we conducted imputation, HLA typing, and sequencing in three independent populations for a total of 689 cases and 2,446 controls. We identified a hexa-allelic amino acid polymorphism at position 13 of the HLA-DR beta chain that showed the strongest association with FL within the major histocompatibility complex (MHC) region (multiallelic p = 2.3 × 10⁻¹⁵). Out of six possible amino acids that occurred at that position within the population, we classified two as high risk (Tyr and Phe), two as low risk (Ser and Arg), and two as moderate risk (His and Gly). There was a 4.2-fold difference in risk (95% confidence interval = 2.9-6.1) between subjects carrying two alleles encoding high-risk amino acids and those carrying two alleles encoding low-risk amino acids (p = 1.01 × 10⁻¹⁴). This coding variant might explain the complex SNP associations identified by GWASs and suggests a common HLA-DR antigen-driven mechanism for the pathogenesis of FL and rheumatoid arthritis.

The Transcriptional Profile of Follicular Lymphoma Associated Regulatory T-Cells (Tregs) Is More Consistent with Tregs Having a Germinal Center Localization and the Ability to Suppress Follicular Helper T-Cells

AbstractAbstract 1536Recent studies have suggested that regulatory T-cells (Tregs) play a critical role in the pathogenesis of follicular lymphoma (FL). We have previously shown that the Tregs infiltrating FL lymph nodes (FLN Tregs) are quantitatively and qualitatively different than those infiltrating normal and reactive nodes (NLN, RLN, respectively). To gain insight into how such Treg populations differ, and how FLN Tregs may impact FL progression and response to therapy, we performed RNA sequence analyses on flow sorted Tregs (CD3+CD4+CD25brightCD127−) from NLN (n=10), FLN (n=12) and RLN (n=7).Using the RefSeq annotated set of mRNA we first identified 11686 annotated genes that were expressed in each of the Treg groups. Using pairwise unequal variance t-tests at p<0.01, we identified 798 genes that differed between NLN and FLN, 589 that differed between NLN and RLN, and 475 genes that differed between FLN and RLN. The transcriptome profile of the FLN Tregs differed most from that of NLN Tregs, with 281 genes increased in FLN relative to NLN, and 517 genes decreased in FLN Tregs relative to NLN Tregs. In contrast, 404 genes were increased and 70 genes decreased in RLN Tregs relative to FLN Tregs suggesting that differences in the transcriptome profile between NLN and FLN Tregs are due, in part, to inflammatory signals shared between FLN and RLN. Tregs, which express FOXP3, are a heterogeneous population which share a “core suppressive module” of CD25 and CTLA-4, but which adapt to microenvironmental signals by co-opting/or up-regulating the canonical transcription factor (TF) of the T-cell population the Treg needs to suppress. This results in modulation of the Treg chemokine receptors in such a fashion that allows these Tregs to traffic to and suppress the specific T-cell population defined by their TF expression. Relative to NLN, FLN Tregs have similar expression of FOXP3 and CD25 but greater expression of CTLA-4 (confirmed at the protein level), which may account, in part, for the greater suppressive activity of FLN Tregs compared to NLN Tregs. Expression of other genes that differ between NLN and FLN which encode for proteins that modulate Treg suppression include IL-10 (up in FLN relative to NLN), S1PR1 and ENTPD1 (both down in FLN relative to NLN), which all favor greater suppression by FLN Tregs. In contrast LGALS1 and NT5E expression (both down in FLN relative to NLN) would favor less suppression by FLN Tregs. Relative to NLN, FLN Tregs have greater BCL-6 expression, consistent with a population that suppress follicular helper T-cells (Tfh), which support normal germinal center (GC) and FL-B cell survival. Consistent with their increased expression of BCL-6, FLN Tregs have greater expression of CXCR5, CXCR4 and CXCL13 and decreased expression of CCR7 compared to that of NLN Tregs, a repertoire associated with GC trafficking. The significant lower expression of both S1PR1 and SELL (CD62L; p<0.000002, and fold changes of −9.0 and −2.8, respectively) that is found in FLN Tregs compared to NLN Tregs is consistent with the greater nodal retention. The lower expression of RORc found in the FLN Tregs would predict for decreased IL-17 secretion and their elevated levels of STAT3 which would support the ability of the FLN Tregs to inhibit Th17 cells.In conclusion, the comparison of the transcriptome profiles of FLN and NLN Tregs; a) suggest that the observed differences are shaped, in part, by inflammatory signals also present in RLN; b) lay the foundation of a hypothesis to explain the molecular basis for their greater immunosuppressive capacity; and c) suggest that FL Tregs are ‘poised' to localize in the GC, be strongly retained in the lymph node and have enhanced potential to suppress Tfh and Th17 cells. Such findings offer new insights into how FL Tregs may contribute to the biology of these B cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Phase II Study of Enzastaurin in Patients with Follicular Lymphoma: Updated Final Clinical Results and Immunohistochemical Correlations

AbstractAbstract 777 Background:Protein kinase C β (PKCβ) is critical for B-cell signaling and survival. Over-expression of PKCβ is implicated in the pathogenesis of follicular lymphoma (FL). Enzastaurin (ENZ), an oral serine/threonine kinase inhibitor, targets the PKCβ and PI3K/AKT pathways to inhibit tumor cell proliferation, induce apoptosis, and suppress tumor-induced angiogenesis. Reported here are the final clinical and correlative study results from a phase II study. Methods:This was an open-label, single-arm, study of patients (pts) with hiastologically confirmed grade 1/2 and stage III/IV measurable FL. Pts were either chemonaive or relapsed after 1 chemotherapy regimen or single-agent rituximab. Pts received 500 mg oral ENZ once daily (1125-mg loading dose on D1) ≤ 3 years or until progression, withdrawal, or toxicity. The primary endpoint was response rate (RR). Secondary objectives included progression-free survival (PFS), duration of response (DoR), and safety. Pre-treatment samples were used for correlative research. Staining was performed for BCL2, BCL6, CD10, Ki67, FOXP1, FOXP3, CD20, MUM1, CD68, and PD1 expression. ENZ-specific biomarkers investigated by immunohistochemistry (IHC; standard H scores range 0–300) included PKCβ2, PTEN, pS6, pGSK3β, and pCREB. Logistic/Cox regression determined the statistical correlation between clinical outcomes (RR or PFS) and IHC for each marker. All tests of association were conducted at a 2-sided α = 0.05. Clinical Results:Sixty-six pts (23 male, 43 female; median age 62 years 3382) were enrolled. Based on FL International Prognostic Index, 12% had low-risk disease (0-1 risk factors [RF]); 55%, intermediate-risk disease (2 RFs); and 33%, high-risk disease (3-5 RFs). Forty-six pts (69.7%) were chemonaive and 20 pts (30.3%) had ≥ 1 prior chemotherapy. The median overall exposure was 10.8 months (mo; 1–57 mo). Of 58 pts who received ≥1 dose of ENZ, 2 (3.4%) had complete response, and 15 (25.9%) had partial response (overall RR = 29.3%). The median PFS was 15.2 mo (95% confidence interval [CI] 11.1–25.7 mo). DoR median was 20.5 mo (95% CI 9.1–31.5 mo). Seven pts (10.6%) discontinued treatment due to adverse events (AEs), of which 5 (7.6%) were possibly drug related: bronchitis, prolonged QTc interval, abdominal distention, colitis, and skin rash. Four pts (6.1%) had drug-related serious AEs: grade (Gr) 3 bronchitis, Gr 2 vision, Gr 2 diarrhea, Gr 2 pancreatitis. There were no drug-related deaths. To date, 5 pts remain on treatment and have been on treatment 3.5–5 years. Correlative Results:Tumor tissue was obtained from 28 pts. Patient demographics and overall RR were well balanced between protocol and correlative study populations. No significant associations with RR or PFS were observed for any FL-specific markers. Significant associations were observed between low cytoplasmic PKCβ2 and increased RR (median H score cutpoint 151.5, odds ratio (OR) [95%CI] = 0.027 [0.001-0.656], p=0.027) (Table 1, Figure 1). Significant associations were also observed between PFS and both cytoplasmic PKCβ2 and cytoplasmic pS6 using median H score cutpoint (Table 1). Conclusions:ENZ has clinical activity and is well tolerated in pts with grade 1/2 FL. Low cytoplasmic PKCβ2 was significantly associated with increased RR and longer PFS; high cytoplasmic pS6 was significantly associated with longer PFS. The correlative results are hypothesis generating given the study’s size and nonrandomized nature. [Display omitted] [Display omitted] Disclosures:Hermann:Eli Lilly and Company: Research Funding. Hsi:Eli Lilly and Company: Research Funding. Hamid:Eli Lilly and Company: Employment. Myrand:Eli Lilly and Company: Employment. Lin:Eli Lilly and Company: Employment. Thornton:Eli Lilly and Company: Employment. Shi:Eli Lilly and Company: Employment. Nguyen:Eli Lilly and Company: Employment.

Pathogenesis of follicular lymphoma

The hallmark t(14;18)(q32;q21) in follicular lymphoma (FL) results in constitutive overexpression of the BCL2 protein, allowing B cells to abrogate the default germinal center apoptotic program. Most tumors are characterized by recurrent secondary genetic alterations including genomic gains, losses, and mutations, some providing a growth advantage, including alterations in MLL2, EPHA7, TNFRSF14, and EZH2. The sequence in which these events occur and how they contribute to progression and ultimately to transformation is unclear. Lastly, crosstalk between neoplastic B cells and non-neoplastic immune and stromal cells in the microenvironment plays an important role in sustaining tumor cell growth, cultivating immune privilege, and promoting transformation.