Follicular Lymphoma Samples Research Articles

IBCL-271 Primary Efficacy and Safety Analysis of a Global Phase II Study of Zandelisib Administered by Intermittent Dosing (ID) in Patients With Relapsed or Refractory (R/R) Follicular Lymphoma (FL): The TIDAL Study

Zandelisib is a selective PI3Kδ inhibitor administered orally at 60 mg once daily (QD) for 2 cycles (response induction), then intermittent dosing (ID) on days 1-7 of subsequent 28-day cycles for maintenance, while potentially enabling regulatory T-cell recovery to reduce risk of immune adverse events (irAEs) seen with continuous PI3Kδ inhibition. In a phase Ib study of zandelisib in 37 R/R FL patients, the overall response rate (ORR) was 87% (78% single agent; 95% with rituximab), with only 8% discontinuations due to irAEs (Pagel ICML 2021). Topline results from the fully enrolled FL population from TIDAL, a global phase II study evaluating zandelisib in R/R indolent lymphomas (NCT03768505). Age ≥18y with FL Grade I-IIIA, progressive disease after ≥2 prior therapies, and no prior PI3K inhibitor. Consent required. FL sample size (planned): 120 patients. Primary efficacy population (PEP): first 91 patients treated. Zandelisib 60 mg QD for 2 cycles, followed by ID during cycle 3+. IRC-assessed ORR (Lugano criteria) after a minimum 6-month follow-up. 91 FL patients in PEP (of 121 enrolled): median 3 prior therapies (range, 2-8), 21 (23%) prior stem cell transplant, 42 (46%) refractory to last therapy, 31 (34%) tumors ≥5 cm, 51 (56%) POD24. ORR was 70.3% (64/91; 95% CI: 59.8%, 79.5%) and complete response (CR) rate was 35.2% (32/91; 95% CI: 25.4%, 45.9%). Responses occurred early: 87.5% (56/91) occurred at end of Cycle 2, 75% (24/91) of CRs at end of Cycle 4. Data still immature for accurate duration of response (DOR) estimation. With median follow-up of 9.4 months (range, 0.8-24) for all 121 patients, 12 (9.9%) discontinued due to any treatment-related AE. Grade 3 AEs of special interest (AESI) included diarrhea (6/121; 5%), colitis (2/121; 1.7%), rash (4/121; 3.3%), stomatitis (3/121; 2.5%), and AST and ALT elevation and non-infectious pneumonitis (1/121 each; 0.8%). Most Grade 3 AESIs (15 [83%]) occurred during daily dosing (cycles 1-3). Zandelisib on ID led to high ORR and CR rates in heavily pretreated FL patients and was associated with a low rate of grade 3 AESI and discontinuations due to treatment-related AEs.

Efficacy and safety of zandelisib administered by intermittent dosing (ID) in patients with relapsed or refractory (R/R) follicular lymphoma (FL): Primary analysis of the global phase 2 study TIDAL.

7511 Background: Zandelisib, a PI3Kδ inhibitor with high target-binding affinity, is administered by intermittent dosing (ID) on days 1-7 of 28-day cycles to potentially enable regulatory T-cell repopulation and lower the risk of immune adverse events (irAEs) seen with continuous PI3Kδ inhibition. In a phase 1b study in 37 patients (pts) with R/R FL, zandelisib daily for two 28-day cycles for tumor debulking then on ID achieved an overall response rate (ORR) of 87% (78% single agent and 95% with rituximab) with < 10% irAEs (Pagel et al. ICML 2021; #113). We conducted the TIDAL study to further evaluate zandelisib in R/R indolent lymphomas (NCT03768505). Methods: Eligible pts ≥18 years with FL Grade I-IIIA, progressive disease after ≥2 prior therapies, and no prior PI3K inhibitor, gave consent and received zandelisib 60 mg daily for 2 cycles then on ID. Not reported here are an arm randomizing pts to zandelisib daily continuously, closed to enrollment early, and an actively enrolling arm in R/R marginal zone lymphoma (MZL). The planned FL sample size was 120 pts on ID, with the primary efficacy population (PEP) pre-defined as the first 91 pts treated. The primary efficacy endpoint was ORR by the Lugano criteria in the PEP as assessed by independent review and analyzed after a minimum 6-month follow-up. Results: 121 FL pts were enrolled. In the PEP (N = 91), the median number of prior therapies was 3 (range 2-8), 21 pts (23%) had received prior stem cell transplant, 42 (46%) had disease refractory to last therapy, 31 (34%) had tumors ≥5 cm, and 51 (56%) were POD24. Overall response rate was 70.3% (n = 64) (95% CI 59.8-79.5%) and complete response rate was 35.2% (n = 32) (95% CI 25.4-45.9%). Responses occurred early with 87.5% (n = 56) of responses observed at the end of Cycle 2 and 75% (n = 24) of CRs at the end of Cycle 4. The data are still immature to estimate accurately the duration of response (DOR). With a median follow-up of 9.4 months (range 0.8-24) for all 121 pts, 12 pts (9.9%) discontinued therapy due to any drug-related AE. Grade 3 AEs of special interest (AESI) were diarrhea in 6 pts (5%), colitis in 2 (1.7%), rash in 4 (3.3%), stomatitis in 3 (2.5%), and 1 (0.8%) each for AST and ALT elevation, and non-infectious pneumonitis. Grade 3 AESIs primarily (15 of 18, 83%) occurred in cycles 1-3, during daily dosing. Conclusions: Zandelisib on ID achieved high ORR and CR rate in heavily pretreated FL pts, and was associated with < 10% of discontinuations due to drug-related AEs and grade 3 AESI, results comparable to the Phase 1b study. Longer follow-up is needed to estimate median DOR. This profile supports evaluation of zandelisib alone and in combination in various B-cell malignancies, both in relapsed disease and earlier lines of therapy. Zandelisib plus rituximab vs chemoimmunotherapy is being studied in the phase 3 trial COASTAL in R/R FL and MZL (NCT 04745832). Clinical trial information: NCT03768505.

Tumor Microenvironment and Microvascular Density in Follicular Lymphoma.

Follicular lymphoma (FL) is a slowly progressive disease and constitutes the second most common non-Hodgkin lymphoma. Biological factors, such as the tumor microenvironment and the host response, are determinants in the outcome of FL but the experimental data about microenvironment and tumor cells in FL are variable and contradictory. In this morphometric study, we analyzed by immunohistochemistry the cellular components of the tumor microenvironment and correlated these data with the microvascular vascular density in three different grades of FL lymph node biopsies, comparing the results to healthy lymph node controls. The results indicated a significant increase in the number of CD68+ and CD163+ macrophages in all three analyzed FL grades. Tryptase+ mast cells resulted in an increase only in grade 1. PDL-1+ cells, CD4- and CD8-lymphocytes number results were reduced in FL samples. The higher number of CD34+ microvessels in the FL grades 1 and 2 of samples positively correlated with CD68+ and CD163+ cells, underlining the important angiogenic potential of this subset of macrophages.

Distinct clinical and genetic features of hepatitis B virus–associated follicular lymphoma in Chinese patients

AbstractHepatitis B virus (HBV) infection has been associated with an increased risk for B-cell lymphomas. We previously showed that 20% of diffuse large B-cell lymphoma (DLBCL) patients from China, an endemic area of HBV infection, have chronic HBV infection (surface antigen–positive, HBsAg+) and are characterized by distinct clinical and genetic features. Here, we showed that 24% of follicular lymphoma (FL) Chinese patients are HBsAg+. Compared with the HBsAg− FL patients, HBsAg+ patients are younger, have a higher histological grade at diagnosis, and have a higher incidence of disease progression within 24 months. Moreover, by sequencing the genomes of 109 FL tumors, we observed enhanced mutagenesis and distinct genetic profile in HBsAg+ FLs, with a unique set of preferentially mutated genes (TNFAIP3, FAS, HIST1H1C, KLF2, TP53, PIM1, TMSB4X, DUSP2, TAGAP, LYN, and SETD2) but lack of the hallmark of HBsAg− FLs (ie, IGH/BCL2 translocations and CREBBP mutations). Transcriptomic analyses further showed that HBsAg+ FLs displayed gene-expression signatures resembling the activated B-cell–like subtype of diffuse large B-cell lymphoma, involving IRF4-targeted genes and NF-κB/MYD88 signaling pathways. Finally, we identified an increased infiltration of CD8+ memory T cells, CD4+ Th1 cells, and M1 macrophages and higher T-cell exhaustion gene signature in HBsAg+ FL samples. Taken together, we present new genetic/epigenetic evidence that links chronic HBV infection to B-cell lymphomagenesis, and HBV-associated FL is likely to have a distinct cell-of-origin and represent as a separate subtype of FL. Targetable genetic/epigenetic alterations identified in tumors and their associated tumor microenvironment may provide potential novel therapeutic approaches for this subgroup of patients.

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.

Diversity of Intratumoral Regulatory T Cells in Non-Hodgkin Lymphoma

Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 + T cells, represent a key challenge in the tumor microenvironment by limiting potent antitumor immune responses. While high densities of tumor-infiltrating Tregs are associated with poor prognosis in patients with various types of solid cancers, their prognostic impact in B-cell non-Hodgkin lymphoma (NHL) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotype and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Our in-depth characterization of Tregs in NHL tumors could open new paths for rational drug design, facilitating selective therapeutic manipulation of Tregs to reduce immunosuppression and improve anti-tumor immunity.Methods: Single-cell suspensions from NHL patients (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and healthy donors (tonsils and peripheral blood)) were analyzed by fluorescent flow- and mass cytometry to characterize Tregs, focusing on their expression of co-stimulatory and co-inhibitory checkpoint receptors. The immunosuppressive capacity of Tregs was measured by in vitro co-culture of FACS-sorted subsets of Tregs together with autologous CellTrace Violet-labelled T effector cells as responder cells, using samples from FL and tonsils. Live CD4 + T cells were obtained by FACS sorting from DLBCL (n = 3), FL (n = 3) and healthy donor tonsils (n = 3) and subjected to single-cell RNA sequencing (scRNA-seq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and scTCR-seq by the 10X Genomics platform. The computational framework of CIBERSORTx was used to generate unique signature matrices for the three Treg subsets identified by scRNA-seq, to facilitate validation in separate scRNA-seq cohorts (King, Sci Immunol 2021; Roider, Nat Cell Biol 2020), and to impute frequencies of the Treg subsets in cohorts with bulk RNA-seq data (Chapuy, Nat Med 2018; Schmitz, NEJM 2018; Pastore, Lancet Oncol 2015).Results: Immunophenotyping by mass cytometry revealed a subset of activated Tregs identified by co-expression of TIGIT, CTLA-4, PD-1, ICOS and OX40, and higher expression of FOXP3, CD25 and CD45RO, that was present in DLBCL and tonsils, but lacking in peripheral blood. This was validated by fluorescent flow cytometry, demonstrating significantly higher frequencies of activated Tregs in NHL tumors compared to PBMCs and tonsils from healthy donors. The phenotypic heterogeneity of intratumoral Tregs reflected different suppressive capacities as activated Tregs more potently suppressed the proliferation of autologous effector CD4 + and CD8 + T cells than naïve Tregs. For global transcriptomic profiling of CD4 + T cells from FL, DLBCL and tonsillar samples, we integrating scRNA-seq and CITE-seq data from 17,774 cells, revealing 13 distinct cellular states including three states of Tregs: naïve, activated and non-conventional LAG3 +FOXP3 - Tregs. Activated Tregs had higher expression of checkpoint receptors (TNFRSF4, TNFRSF18, ICOS), phosphatases (DUSP2, DUSP4), NF-κB pathway (NFKBIA, TNFAIP3, NFKBIZ, REL), chemokine receptors (CXCR4) and transcription factors (JUNB, IRF1, STAT3) as compared to naïve Tregs. We next used a computational approach to develop unique signature matrices for each Treg subset. This approach demonstrated strong concordance between CIBERSORTx estimated cell abundances of the three Treg subsets and the ground truth, and was validated in two external scRNA-seq cohorts. The development of unique signature matrices for Treg subsets facilitated imputation of their frequencies in bulk RNA-seq datasets. These analyses revealed that higher frequency of activated Tregs was enriched in the germinal B cell subtype of DLBCL and was associated with adverse outcome in FL.Conclusion: This study demonstrates that Tregs infiltrating NHL tumors are transcriptionally and functionally diverse and include highly immunosuppressive activated Tregs co-expressing several checkpoint receptors, which distinguish them from peripheral blood Tregs. Activated intratumoral Tregs could hamper clinical responses to checkpoint blockade, and identifying and targeting their vulnerabilities has the potential to improve anti-tumor immune responses. DisclosuresHolte: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Nordic: Membership on an entity's Board of Directors or advisory committees; Nanovector: Membership on an entity's Board of Directors or advisory committees, Other: lectures honorarias; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Alizadeh: Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Roche: Consultancy, Honoraria; Janssen Oncology: Honoraria; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Bristol Myers Squibb: Research Funding.

Characterization of the Immunogenomic Landscape of Follicular Lymphoma

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

Reproducibility of m7-FLIPI Risk Scores in Follicular Lymphoma Using Tumor Biopsies and Blood Specimens

Background: Follicular lymphoma (FL) is clinically and genetically heterogeneous. While most patients experience an indolent disease course, with median survival of over 15 years, a portion of patients experience early relapse and poor outcomes. The m7-FLIPI risk score has been described as a clinicogenetic method to accurately determine which patients are at highest risk for early relapse and poor outcomes (Pastore et al., Lancet Oncology 2015; Vindi et al., Blood 2015). However, molecular characterization of the m7 genes currently relies on tumor biopsies, which require an invasive procedure and may be unavailable. Furthermore, it is unclear whether the m7-FLIPI score is consistent over the course of disease. Circulating tumor DNA (ctDNA) is an emerging noninvasive biomarker for tumor detection in diverse lymphomas including FL. In this study, we evaluated the stability of m7-FLIPI status over multiple FL relapses as well as the utility of ctDNA for classification of patients with FL into high- and low-risk m7-FLIPI categories.Methods: We applied Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq), a capture-based method for ultra-deep sequencing (Newman et al., Nat Med 2014; Scherer et al., Sci Transl Med 2016 ) to identify risk via m7-FLIPI in pretreatment tumor and plasma samples of 29 FL patients. In addition, we retrospectively analyzed whole exome sequencing (WES) data of 37 patients from two FL cohorts (Green et al., PNAS 2015; Bouska et al., Leukemia 2017). We first validated in silico our capture-based method for identification of m7-FLIPI-relevant somatic alterations in 258 patients from the original publication (Pastore et al., Lancet Oncology 2015). We then identified non-synonymous mutations in CREBBP, EZH2, ARID1A, MEF2B, FOXO1, EP300, and CARD11 presentin each tumor and plasma sample. Using both CAPP-Seq and WES data (n = 66), we compared m7-FLIPI risk group concordance between paired diagnostic tumor biopsies, paired diagnostic and relapse tumor biopsies, paired FL and transformed FL (tFL) tumor biopsies, and paired diagnostic tumor and plasma samples. FLIPI was calculated at first FL time-point available.Results: We observed a 95% agreement in m7-FLIPI risk category between our capture-based method and methods utilizing full exon coverage. A 100% concordance of m7 gene profile and 100% concordance of m7-FLIPI risk category were seen when comparing paired FL tumor biopsy samples from two separate lymph nodes obtained at diagnosis (n = 5). In addition, a 75% agreement in m7 gene profile and 100% agreement of m7-FLIPI risk category were observed when comparing tumor biopsy samples obtained at FL diagnosis and first relapse (n = 36; median time to relapse of 547 days, range 55 - 4200 days). There was a low concordance in m7 genetic profile between tumor biopsy samples from time of initial diagnosis and transformation (48.4%, n = 31). Importantly, concordance of m7 gene profile and m7-FLIPI risk category was 64.3% and 78.6%, respectively, when assessed by paired tumor and plasma samples obtained at FL diagnosis (n = 14; Figure 1). Furthermore, non-concordant mutations could be present in either the tumor or the plasma sample. Across all FL samples, there was no significant difference in concordance based on individual m7 genes. When analyzing pairs in which mutations were observed, concordance between paired samples was lowest for EZH2 (p = 0.028).Conclusions: m7-FLIPI scores appear consistent between multiple FL tumor samples obtained at the time of diagnosis and across FL relapses. However, concordance in m7 gene profile between paired plasma and tumor samples is lower than between paired tumor biopsies, even at ultra-deep sequencing depths. Optimization of blood samples for assessing m7 gene profile should be performed to improve the reliability of ctDNA for determining m7-FLIPI risk status.Figure 1. Concordance of m7-FLIPI risk category. (A) Re-classification of m7-FLIPI risk category when using paired tumor biopsy and plasma samples from 14 patients. (B) Percentage of discordant samples when comparing diagnostic tumor pairs (n = 5), diagnostic versus relapse tumor pairs (n = 37), and diagnostic tumor versus plasma pairs (n = 14). Concordance was lowest for diagnostic tumor and diagnostic plasma pairs (p = 0.013). [Display omitted] DisclosuresDiehn:Varian Medical Systems: Research Funding; Quanticel Pharmaceuticals: Consultancy; Roche: Consultancy; Novartis: Consultancy. Alizadeh:Gilead: Consultancy; Roche: Consultancy; Genentech: Consultancy; Celgene: Consultancy; CiberMed: Consultancy.

Idelalisib Interferes with the Crosstalk of Follicular Lymphoma and Its Immune Microenvironment and Potentiates the Activity of ABT-199

Background:The PI3K pathway is crucial in B-cell non-Hodgkin lymphoma (NHL) cell survival by regulation of cell cycle, apoptosis, proliferation, differentiation, homing, and retention. Idelalisib (IDELA/GS-1101/CAL-101) is a first-in-class PI3Kδ selective inhibitor that is approved for the treatment of relapsed chronic lymphocytic leukemia and follicular lymphoma (FL). The mechanisms of IDELA sensitivity and resistance remain to be fully elucidated.We aimed to characterize the cellular and molecular effects of IDELA in an in vitro model utilizing a primary FL cell co-culture system with follicular dendritic cells (FDC) or M2-polarized macrophages (M2), both subpopulations related to poor prognosis, in order to explore possible combinatorial regimens.Methods: FL primary cells from lymph node biopsies were co-cultured (or not) (24-72h) with FDC cells or M2 macrophages. Viability of FL cells was assessed by flow cytometry (AnnexinV/7-AAD). B cells were isolated using CD20 magnetic beads, RNA extracted and subjected to gene expression profiling (GEP), and data analyzed by Gene Set Enrichment Analysis (GSEA).Results: In the absence of accompanying cells, IDELA (100-500nM) induced a moderate cytotoxic effect on FL cells (n=10) that was maintained in FL-FDC and FL-M2 co-cultures. GEP analysis indicated that IDELA (500nM, 48h) induced a downregulation of genes related to germinal center (GC) program, CD40L, and NF-kB pathways, together with other pathways related to B cell-T cell crosstalk. In this sense, IDELA treatment reduced the expression on B-cells of key molecules such as ICOSL (TFH activation) and CCL22 (Treg activation).In the context of FL-FDC co-culture, FDC induced the transcription of genes related to B-cell receptor-, CD40- and NF-kB-signaling pathways, GC program, mechanistic target of rapamycin (mTOR), angiogenesis, extracellular matrix remodeling, and migration, among others. IDELA blocks the activation of the mTOR pathway in all FL samples analyzed (n=4), but displayed differential activity on the rest of the pathways. We are currently investigating the molecular basis of these selective molecular responses to IDELA in vitro and its possible correlation with the presence of somatic mutations in FL (CREBBP, EP300, EPHA7, EZH2, KMT2D, MEF2B, RRAGC, TNFAIP3 and TNFRSF14).We have also explored the combination of IDELA with the B-cell lymphoma 2 (BCL2) inhibitor ABT-199/Venetoclax and have uncovered a synergistic interaction between the two drugs that is maintained in FL-FDC/M2 co-cultures (n=9). This synergy may be linked to the downregulation of the anti-apoptotic proteins Mcl-1, Bcl-XL, and BFL-1 induced by IDELA, together with the induction of the pro-apoptotic BH3-only HRK and BIM. We are currently performing BH3 profiling of FL primary cellswith FDC-M2 co-cultures to identify which patients may be more susceptible to this drug combination.Conclusions: Our results suggest a differential role of IDELA in the context of FL-FDC/FL-T cell crosstalk that may be linked to the mutational patient profile. IDELA ABT-199 combination treatment is highly efficacious in FL-FDC/M2 co-cultures. DisclosuresYahiaoui:Gilead Sciences, Inc.: Employment, Equity Ownership. Lopez-Guillermo:Janssen: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Roche: Consultancy, Other: Research grant; Novartis: Consultancy. Roue:Gilead Sciences, Inc.: Research Funding. Tannheimer:Gilead Sciences, Inc.: Employment. Pérez-Galán:Gilead Sciences, Inc.: Research Funding.

Follicular Lymphoma Regulatory T Cell Function and Origin

Follicular Lymphoma (FL) is the most frequent indolent lymphoma and results from the malignant transformation of germinal center (GC) B cells. Among FL recurrent genetic alterations, some are not only oncogenic per se but favor the interactions of FL B cells with their microenvironment including in particular surrounding follicular helper T cells (Tfh). During normal GC reaction Tfh support early GC B-cell selection and differentiation whereas follicular regulatory T cells (Tfr) expand later and inhibit both Tfh activity and B-cell antibody secretion. Whereas the role of Tfh in human diseases is an intense matter of interest, very few data are available concerning the origin and function of human Tfr. We previously demonstrated that Tfh and Tfr proportions are increased within FL lymph nodes and FL-Tfh contribute, through a specific polarization profile, to FL B cell survival and drug resistance. Interestingly FL-Tfr expansion is correlated to the one of FL-Tfh but not FL-Treg. The aim of this work was to characterize human Tfr in non-malignant (chronically inflamed tonsils) and malignant (FL) settings through transcriptomic, DNA methylation, TCR repertoire, and functional studies. We demonstrated for the first time that human Tfr purified from FL samples and tonsils directly inhibit autologous Tfh proliferation and trigger a downregulation of the co-stimulatory markers CD86 and HLA-DR on autologous normal and malignant GC B cells. In agreement FL-Tfh proliferated less than tonsil-Tfh in situ and after in vitro restimulation. Moreover, we found that Tfr clustered with Treg at both transcriptomic and methylome levels including for the high demethylation level of the FOXP3 TSDR, suggesting a stable regulatory phenotype. However, TCR sequencing demonstrated that tonsil-Tfr shared a common repertoire only with tonsil-Tfh unlike tonsil-Treg, arguing for their peripheral unlike thymic origin. Conversely in FL, even if the majority of common clones were found between Tfr and Tfh, a small proportion of TCR repertoire was shared between Tfr and Treg. Altogether, these results are the first detailed characterization of human Tfr and challenge our current understanding of Tfr commitment pathway. FL-Tfr retain their capacity to inhibit Tfh thus questioning their role in FL and their potential as therapeutic target in this fatal malignancy. DisclosuresTarte:Roche: Consultancy; Novimmune: Research Funding; Celgene: Consultancy, Research Funding. Lamy:Roche: Consultancy, Honoraria.

Lack of stem-like exhausted T cells and increased inhibitory receptor expression distinguishes diffuse large B cell lymphomas from Hodgkin lymphoma.

Abstract Immune checkpoint (IC) blockade has revolutionized the treatment of chemo-refractory solid tumors and has demonstrated promising results for the treatment of resistant blood cancers, including lymphoma. However, clinical response to PD1 blockade depends on the subtype of lymphoma, with durable responses observed in 70 % of Hodgkin lymphoma (HL) versus only 10 to 40% of Diffuse Large B-Cell Lymphoma (DLBCL) and Follicular Lymphoma (FL). The biological aspects that hinder the efficacy of immunotherapy in DLBCL and FL are not well-known. Since DLBCL and FL are more refractory to IC therapy than HL, we hypothesized that TILs from DLBCL and FL present a more advanced state of exhaustion than TILs from HL. We performed an immunophenotyping of TILs from 57 HL, FL or DLBCL lymphoma samples collected at diagnosis and frozen as viable cell suspension. Our data revealed that CD8 TILs from DLBCL express higher levels of ICs (PD1, Tim3 and Lag3) compared to TILs from FL and HL. Boolean analysis demonstrated that DLBCL and FL had a higher frequency of CD8T cells that co-expressed 6–7 ICs, compared to HL, suggesting more advanced exhaustion. In contrast, we observed that HL and FL exhibited significantly more stem-like exhausted cells (PD1+/Tim3−/CD38−/TCF1+) compared to DLBCL. Stem-like exhausted CD8 T cells are driven by the transcription factor TCF-1 and extensively proliferate in response to PD1 blockade. Collectively, our data indicate that TILs from DLBCL and FL express a whole array of ICs. DLBCL further lack protective stem-like CD8 T-cell subsets which correlate with poor clinical outcome. These results strengthen our understanding of the cellular defects precluding potent anti-tumor T cell functions in DLBCL and FL.

Large-Scale Proteomic Analysis of Follicular Lymphoma Reveals Extensive Remodeling of Cell Adhesion Pathway and Identifies Hub Proteins Related to the Lymphomagenesis.

Simple SummaryFollicular lymphoma represents the major subtype of indolent B-cell non-Hodgkin lymphomas, ranging from about 20 to 30% of all B-NHLs cases in western countries. Yet, the global proteome profile of follicular lymphoma remains largely undocumented; thus, we aimed to employ for the first time a comprehensive proteomic analysis to outline its molecular landscape. A total of 15 lymphoma fine-needle aspiration biopsy samples and 14 controls were evaluated by label-free quantitative proteomics. Among the 7673 proteins identified in our dataset, 1186 proteins were differentially expressed between lymphoma and control samples. Importantly, dysregulated proteins were enriched in biological processes such as B-cell receptor signaling pathway, cellular adhesion molecules pathway, or membrane trafficking. Additionally, we identified several novel hub proteins related to lymphomagenesis. To summarize, we have determined the molecular characteristics of follicular lymphoma and discovered proteins which may hold potential for biomarkers or therapeutic targets.Follicular lymphoma (FL) represents the major subtype of indolent B-cell non-Hodgkin lymphomas (B-NHLs) and results from the malignant transformation of mature B-cells in lymphoid organs. Although gene expression and genomic studies have identified multiple disease driving gene aberrations, only a few proteomic studies focused on the protein level. The present work aimed to examine the proteomic profiles of follicular lymphoma vs. normal B-cells obtained by fine-needle aspiration biopsy (FNAB) to gain deep insight into the most perturbed pathway of FL. The cells of interest were purified by magnetic-activated cell sorting (MACS). High-throughput proteomic profiling was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and allowed to identify of 6724 proteins in at least 75% of each group of samples. The ‘Total Protein Approach’ (TPA) was applied to the absolute quantification of proteins in this study. We identified 1186 differentially abundant proteins (DAPs) between FL and control samples, causing an extensive remodeling of several molecular pathways, including the B-cell receptor signaling pathway, cellular adhesion molecules, and PPAR pathway. Additionally, the construction of protein–protein interactions networks (PPINs) and identification of hub proteins allowed us to indicate the key player proteins for FL pathology. Finally, ICAM1, CD9, and CD79B protein expression was validated in an independent cohort by flow cytometry (FCM), and the results were consistent with the mass spectrometry (MS) data.

PD-1 Expression in Pre-Treatment Follicular Lymphoma Predicts the Risk of Subsequent High-Grade Transformation.

PurposeFollicular lymphoma (FL) is an indolent, yet generally incurable neoplasia with a median survival exceeding 10 years. However, a subset of FL patients experiences histological transformation (HT) to a more aggressive lymphoma, in the majority of cases to diffuse large B-cell lymphoma (DLBCL). This affects both the clinical course and the prognostic outcome, resulting in a markedly reduced survival after transformation. Thus, early risk stratification and prediction of patients at risk of HT would be highly valuable in the clinical setting. Here, we investigated the potential of the immune inhibitory programmed death 1 (PD-1) receptor as a biomarker predictive of HT.Patients and MethodsImmunohistochemical staining and quantification by digital image analysis of PD-1 was performed on diagnostic tumor-tissue samples from FL patients with and without subsequent transformation (n=34 and n=46, respectively), and on paired samples from the transformed lymphoma (n=34).ResultsAt the time of initial FL diagnosis, samples from patients with subsequent HT had significantly higher tumor-tissue expression of PD-1 compared with diagnostic FL samples from patients without subsequent HT (p=0.010). At the time of transformation, PD-1 expression was significantly reduced (p<0.001). No difference was observed in intra-follicular PD-1 expression at FL diagnosis between samples from patients with or without HT; however, high intra-follicular levels of PD-1 were associated with significantly shorter transformation-free survival times (p<0.043).ConclusionOur data suggest that pre-treatment tumor-tissue PD-1 expression already predicts the risk of subsequent transformation to DLBCL, as early as the time of FL diagnosis.

Baseline SUVmax is related to tumor cell proliferation and patient outcome in follicular lymphoma.

Follicular lymphoma (FL) is the most common indolent lymphoma. Despite the clear benefit of CD20-based therapy, a subset of FL patients still progress to aggressive lymphoma. Thus, identifying early biomarkers that incorporate positron emission tomography metrics could be helpful to identify patients with a high risk of treatment failure with rituximab. We retrospectively included a total of 132 untreated FL patients separated into training and validation cohorts. Optimal threshold of baseline whole-body maximum standardized uptake (SUVmax) was first determined in the training cohort (n=48) to predict progression-free survival (PFS). The PET results were investigated along with the tumor and immune microenvironment, which were determined by immunochemistry and transcriptome studies involving gene set enrichment analyses and immune cell deconvolution, together with the tumor mutation profile. We report that baseline SUVmax>14.5 was associated with poorer PFS than baseline SUVmax≤14.5 (hazard ratio =0.28; P=0.00046). Neither immune T-cell infiltration nor immune checkpoint expression were associated with baseline PET metrics. By contrast, FL samples with Ki-67 staining ≥10% showed enrichment of cell cycle/DNA genes (P=0.013) and significantly higher SUVmax values (P=0.007). Despite similar oncogenic pathway alterations in both SUVmax groups of FL samples, four out of five cases harboring the infrequent FOXO1 transcription factor mutation were seen in FL patients with SUVmax>14.5. Thus, high baseline SUVmax reflects FL tumor proliferation and, together with Ki-67 proliferative index, can be used to identify patients at risk of early relapse with rituximab chemotherapy.

Systematic Evaluation of Somatic Cis-Regulatory Mutations in Follicular Lymphoma

Background: Follicular lymphoma (FL) is an incurable indolent B cell malignancy characterized in the majority of cases by the t(14;18) translocation. While the mutational landscape of the coding genome is nearing completion, less is known about the characteristics of its noncoding genome. Our expectation is that the distribution of noncoding mutations will be non-random, determined by epigenomics features, such as chromatin modification and accessibility. Our strategy is therefore to integrate whole genome epigenomic marks and mutations in order to enrich for variants with regulatory potential and identify unique mutational processes specific to thesecis-regulatory elements (CREs).Allele-specific expression (ASE) patterns allow further refinement to resolve functional CREs and bona fide mutations associated with changes in gene expression. Methods: The H3K27Ac consensus CREs were determined using the ChIP-Seq data of 9 FL patient primary cells (Koues et al., Immunity2015). DNase I-hypersensitive sites (DHSs) and Hi-C of the B-lymphocyte cell line GM12878 was downloaded from ENCODE. Whole genome sequencing and RNA-Seq of 70 FL patients with relative high tumour cellularity (≥30% for DNA tumour purity and ≥25% for RNA B-cell content) were obtained from the International Cancer Genome Consortium project ICGC MMML-Seq. The mutation rate within CREs and DHSs in comparison to their flanking sequences was estimated using a previous pipeline (Sabarinathan et al., Nature2016). We further developed cis-ASE, an integrated analytic pipeline for the identification of recurrent ASE genes, and significantly associated CREs and mutations (Fig1.A). Results: In total 1.04 million noncoding mutations, corresponding to 14.8K mutations per sample with a median of 9,991 noncoding mutations were identified in our series of 70 FL samples. 62K (6.0%) mutations were located within H3K27Ac bound CREs and there was an elevated mutation rate in H3K27Ac CREs compared to the corresponding flanking regions of 1kb up and downstream (χ² test p = 1.95e-19, Fig1.B). For DHSs, we observed the opposite pattern, with a lower mutation rate in DHSs than in the flanking regions (p = 0.04, Fig1.C), most likely reflecting the higher accessibility to global genome repair machinery in DHSs in relation to flanking sequences. Dividing mutations into high (≥0.3) and low (&amp;lt;0.3) adjusted variant allele frequency (VAF) groups (accounting for tumour purity), we observed significantly higher mutation rate in CREs than in the flanking sequences, that was specific for mutations with higher VAFs (p= 6.95e−38), as the difference was much weaker for low VAF mutations (p= 0.02). Mutation signatures 6 and 20, linked to defective DNA mismatch repair, were highly enriched for mutations within H3K27Ac CREs (44%) versus other regions outside (27%) (p&amp;lt; 0.001). ASE was assessed using 45.6k informative SNPs (1.7%) per sample wherein matched genotype data was available from WGS and RNA-Seq profiles. Cis-ASE identified on average 480 ASE genes per sample (binomial test, adjusted p&amp;lt;0.05), corresponding to 1,943 recurrent ASE genes with a minimum threshold of ≥5 samples. These ASE events were not significantly influenced by local copy number changes or promoter methylation. KEGG pathway analysis of recurrent ASE genes identified adherens junction, B cell receptor signaling pathway and Fc gamma R-mediated phagocytosis as the most overrepresented pathways. Cis-ASE further identified 18 ASE-CRE interactions where CRE mutations were significantly correlated with an imbalance in elevated alternative allele ratios. These ASE genes included recognized lymphoma related genes including BCL2, STAT6, MAF, and additional novel targets. The pattern of aberrant somatic hypermutation (aSHM) was assessed for these 18 CREs, and we narrowed down to 15 significant ASE-CRE interactions not strongly affected by aSHM, consisting of 92 bona fide mutation candidates present in 37 FL samples of our cohort (52.9%). Conclusion: Our study identified unique mutation processes operating in H3K27Ac CREs. Using an integrated genomic approach of whole genome mutations, chromatin marks and RNA-Seq, we explored ASE-CRE interactions, and identified 15 H3K27Ac bound CREs enriched for cis-regulatory mutations significantly associated with ASE and total expression of targeted genes, deserving for further exploration to enrich our understanding of FL noncoding genome. Disclosures Okosun: BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Research Funding.

Identification of LAG3+ T Cell Populations in the Tumor Microenvironment of Classical Hodgkin Lymphoma and B-Cell Non-Hodgkin Lymphoma

Identification of LAG3+ T Cell Populations in the Tumor Microenvironment of Classical Hodgkin Lymphoma and B-Cell Non-Hodgkin Lymphoma

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

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

Abstract IA13: Oncogenic Rag GTPase signaling links cellular nutrients with the FL microenvironment

Abstract During the humoral response, B cells undergo a sudden anabolic shift that requires high cellular nutrient levels to sustain the subsequent proliferative burst. Follicular lymphoma (FL) originates from B cells that have participated in the humoral response, and 15% of FL samples have selected for point, activating mutations in RRAGC, a member of the Rag GTPase family that controls the activation of the mechanistic target of rapamycin complex 1 downstream of the sensing of cellular nutrients. S74C and T89N, two of the most frequent activating single-amino acid changes in RRAGC, when targeted to the endogenous Rragc locus in mice, confer only a partial insensitivity to nutrient deprivation but strongly exacerbate B-cell responses and accelerate lymphomagenesis. Surprisingly, this moderate increase in nutrient signaling affected the interaction of B cells with the cellular microenvironment, synergizing their effects on mTORC1 activation with paracrine cues from the supportive T-cell microenvironment that activate B cells via the PI3K–Akt–mTORC1 axis. Hence, Rragc mutations sustain induced germinal centers and murine and human FL in the presence of decreased T-cell help. From a therapeutic standpoint, Rragc mutations impose a selective vulnerability to pharmacologic inhibition of mTORC1. Our results support a model in which activating mutations in the nutrient signaling pathway foster lymphomagenesis by corrupting a nutrient-dependent control over paracrine signals from the T-cell microenvironment. While pharmacologic inhibition of mTORC1 with rapamycin yielded exciting preclinical responses in murine lymphomas with activating mutations in Rragc, targeting the nutrient signaling cascade itself, instead of using such allosteric, partial inhibition of mTOR, may constitute a more efficacious intervention. Because nutrient signaling inhibitors are still in development phase, their efficacy and safety remain unproven. Previous genetic approaches to investigate the consequences of inhibition of Rag GTPase signaling relied on deletion of the Rags in mice and led to severe phenotypes and death. Incomplete inhibition of nutrient signaling, an approach that would mirror more closely the effect of small molecules, has not been pursued to support both their efficacy and safety. We have generated knock-in mice endogenously expressing a point-mutant form of RagC (Q119L) that partially suppresses nutrient signaling. RagCQ119L/Q119L mice are not viable, but RagCQ119L/+ mice show minimal phenotypic alterations with partially decreased nutrient signaling. While B-cell development was unaffected, B-cell activation and the humoral response were impaired in RagCQ119L/+ in a B cell-intrinsic manner. When bred to the FL- and autoimmunity-prone strain VavP-Bcl2, RagCQ119L/+ mice were protected against development of both FL and autoimmunity. No obvious systemic tradeoff for the suppression of nutrient signaling seems to occur, because RagCQ119L/+ mice show normal physiology and longevity with a similar age-dependent health decline. Altogether, our work supports the oncogenicity of activating mutations in components of the nutrient signaling pathway, such as RagC, and both the efficacy and safety of a moderate inhibition of nutrient signaling against pathologic B cells without detrimental systemic effects. Citation Format: Ana Ortega-Molina, Cristina Lebrero-Fernández, Nerea Deleyto-Seldas, Alba Sanz, Alejo Efeyan. Oncogenic Rag GTPase signaling links cellular nutrients with the FL microenvironment [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA13.

PI3Kδ inhibition reshapes follicular lymphoma–immune microenvironment cross talk and unleashes the activity of venetoclax

AbstractDespite idelalisib approval in relapsed follicular lymphoma (FL), a complete characterization of the immunomodulatory consequences of phosphatidylinositol 3-kinase δ (PI3Kδ) inhibition, biomarkers of response, and potential combinatorial therapies in FL remain to be established. Using ex vivo cocultures of FL patient biopsies and follicular dendritic cells (FDCs) to mimic the germinal center (n = 42), we uncovered that PI3Kδ inhibition interferes with FDC-induced genes related to angiogenesis, extracellular matrix formation, and transendothelial migration in a subset of FL samples, defining an 18-gene signature fingerprint of idelalisib sensitivity. A common hallmark of idelalisib found in all FL cases was its interference with the CD40/CD40L pathway and induced proliferation, together with the downregulation of proteins crucial for B–T-cell synapses, leading to an inefficient cross talk between FL cells and the supportive T-follicular helper cells (TFH). Moreover, idelalisib downmodulates the chemokine CCL22, hampering the recruitment of TFH and immunosupressive T-regulatory cells to the FL niche, leading to a less supportive and tolerogenic immune microenvironment. Finally, using BH3 profiling, we uncovered that FL–FDC and FL–macrophage cocultures augment tumor addiction to BCL-XL and MCL-1 or BFL-1, respectively, limiting the cytotoxic activity of the BCL-2 inhibitor venetoclax. Idelalisib restored FL dependence on BCL-2 and venetoclax activity. In summary, idelalisib exhibits a patient-dependent activity toward angiogenesis and lymphoma dissemination. In all FL cases, idelalisib exerts a general reshaping of the FL immune microenvironment and restores dependence on BCL-2, predisposing FL to cell death, providing a mechanistic rationale for investigating the combination of PI3Kδ inhibitors and venetoclax in clinical trials.

CD4 Lymphocytes and T Regulatory Cells Show Distinct Phenotypes in Follicular Lymphoma and Classical Hodgkin Lymphoma Which May Account for Differences in Responses to Checkpoint Therapy

Physiologically T-regulatory cells (Treg) suppress immune responses against self-antigens preventing autoimmunity. However, in cancer they are believed to suppress anti-tumor immune response, and the presence of Treg in the tumor microenvironment has been associated with adverse outcome in most cancers. Treg are integral part of tumor microenvironment in lymphoma, and can use different mechanisms to inhibit both adaptive and immune response (including CD4, CD8, DC, macrophages, B-cells and NK cells) modulating therefore the interaction between lymphoma and the host microenvironment. Increased numbers of Treg have been associated with adverse clinical outcome in follicular lymphoma (FL) but a more favorable outcome in classical Hodgkin lymphoma (CHL). In this study we examined Treg phenotype in detail using multiparameter flow cytometry in lymph node specimens with normal histology (NLN) and involved by FL and CHL. We report that both CD4 positive T-cells and Treg subset show distinct phenotypes in different disease entities suggesting different biological functions. Single cell suspensions were prepared from NLN (n=10) and lymph node biopsies involved by FL (n=10) and CHL (n=10), and high dimensional multiparameter flow cytometric immunophenotyping was performed using antibodies against immune checkpoints (TIGIT, TIM3, PD-1, CD96, LAG3, CTLA4, CD73). To characterize the CD4 T-cell compartments we used a dimensionality reduction algorithm for non-linear data representations (tSNE). We studied the CD4+FoxP3-CD25- and Treg characterized by CD4+FoxP3+CD25+ phenotype separately and in both the compartments we identified and characterized subpopulations specific for each of the disease cohorts. tSNE representations CD4 positive T-cells and Treg showed different distributions in NLN, FL and HL. In NLN, CD4 cells broader heterogeneity without distinct clusters whereas in FL and CHL CD4 positive T-cells and Treg (Figure 2 A-B) showed highly polarized phenotypes which were distinct from each other and nearly absent within normal Treg compartment. We observed a strong expression of the immune checkpoint regulators TIM3, LAG3, CTL4, Tbet and PD1 in CD4 T-cells derived from HL tissues. In contrast FL CD4 T-cells were mainly characterized by an up regulation of PD1, CTLA4 and TIGIT. The TIGIT+ cells in FL samples are mainly CXCR5+PD1bright and they up regulate CTLA-4 in the FoxP3+ compartment. In all the neoplastic tissues the FoxP3+CD25+ T-Reg express mainly an activated/memory phenotype (CD45RO+CTLA4+), but while in HL microenvironment they show a TH1 phenotype (CXCR3+Tbet+), in FL they mainly express PD1+CXCR5+ (Figure 1B). Additionally, the dominant population of regulatory T-cells derived from HL samples down modulate PD1 expression, show high level of expression of TH1-associated transcription factor Tbet and have high proliferation index, while in FL, PD1 is brightly expressed, Tbet is not expressed and proliferation index is low in the dominant population (Figure 1A). The distinct phenotypic differences of Treg in FL lymphoma and CHL may account for the better prognosis seen in CHL with increased Treg which has TH1 like phenotype, therefore predicted to have anti-tumor activity. The differences seen in the expression immune checkpoint regulators both on CD4 positive T-cells and Treg subset may explain different rate responses seen in FL compared to CHL with checkpoint therapy. Disclosures Roshal: Physicians' Education Resource: Other: Provision of services; Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services. Dogan:Corvus Pharmaceuticals: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Roche: Consultancy, Research Funding; Celgene: Consultancy; Seattle Genetics: Consultancy.