Follicular Lymphoma Tumor Cells Research Articles

Co-Occurrence of Clonally Related Follicular Lymphoma and Histiocytic Sarcoma

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

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

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

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

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

Molecular Pathogenesis of Follicular Lymphoma: From Genetics to Clinical Practice

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

In Situ Vaccination Induces Changes in Follicular Lymphoma Tumor Cells That Correlate with Abscopal Clinical Regressions

In Situ Vaccination Induces Changes in Follicular Lymphoma Tumor Cells That Correlate with Abscopal Clinical Regressions

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 & Royalties. Plotnikova:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Galkin:BostonGene: Current Employment, Patents & Royalties. Postovalova:BostonGene: Current Employment, Current equity holder in private company. Svekolkin:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Isaev:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Lozinsky:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Meerson:BostonGene: Current Employment. Varlamova:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Ovcharov:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Polyakova:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Nomie:BostonGene: Current Employment, Current equity holder in private company. Kotlov:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Tsiper:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Frenkel:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Attaulakhanov:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Fowler:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties.

Clinical significance of tryptophan catabolism in follicular lymphoma

The enzyme, indoleamine 2,3-dioxygenase 1 (IDO), catabolizes tryptophan (Trp) in the kynurenine (Kyn) pathway, and is important in suppressing antitumor immune responses in the tumor microenvironment. With regard to previously untreated patients with follicular lymphoma (FL), we sought to establish the prognostic significance of Trp catabolism in this disease. Serum Trp and Kyn levels in 110 patients with FL were quantified, and their relationship to different clinical parameters studied. IDO expression in the lymph nodes of affected patients was studied. Study participants included 54 males and 56 females (age range 39-86, median 62 years), showing a 5-year overall survival (OS) rate of 78.5%. Patients with a high Kyn level (5-year OS, 65.0% vs. 81.7%; p=0.026), high Kyn/Trp ratio (71.1% vs. 81.7%; p=0.002), and low hemoglobin (Hb) level (<12.0 g/dL; p=0.001; a component of FL international prognostic indexes) demonstrated a significantly shorter OS. Multivariate analysis included the following 10 variables: age, sex, serum β2-microglobulin, Hb, longest diameter of the largest involved node, Ann Arbor stage, serum lactate dehydrogenase, histologic grading, B symptoms, and serum Kyn/Trp ratio; a lower Hb level and a high Kyn/Trp ratio (HR, 3.239; 95% CI, 1.296-8.096) led to a significantly inferior OS. In the microenvironment, some CD11c-positive myeloid dendritic cells but not FL tumor cells were found to produce IDO. Overall, measuring levels of serum Kyn and Trp in individual patients with FL contributed to predicting their prognosis.

Characteristics of Hematopoiesis in Follicular Lymphoma Patients

Aim. To assess hematopoiesis in follicular lymphoma (FL) patients at different disease stages with different morphologic structures of tumor and bone marrow microenvironment. Materials &amp; Methods. The trial included 152 FL patients treated from 2006 to 2016. In all of them the diagnosis was based on immunohistochemical analysis of extramedullar tumor as well as the analysis of bone marrow aspirates and core biopsy samples. In cases of bone marrow lesions (n = 33) a detailed morphoimmunophenotypic evaluation of tumor cells was carried out by means of flow cytometry, and lymphocyte subset panel evaluation was performed. Results. Anemia, thrombocytopenia, and monocytosis in blood of FL patients are not associated with bone marrow lesions. In the absence of signs of these lesions anemia was detected in 23 (19 %) patients, thrombocytopenia was identified in 8 (7 %) patients, and 11 (9.1 %) patients showed monocytosis. Among patients with bone marrow lesions 9 (27.2 %) anemia, 11 (33.8 %) thrombocytopenia, and 7 (21 %) monocytosis cases were reported. Depth of cytopenia was determined by the degree of bone marrow tumor infiltration. Based on lymphocyte subset panel evaluation the following types of tumor cells in bone marrow aspirates were characterized: elements with blastic structure of nuclear chromatin, atypical lymphoid cells, and those similar to normal lymphocytes. Immunophenotypic heterogeneity of tumor cells in bone marrow was demonstrated. The trial showed that hemoglobin level, the count of blood thrombocytes and monocytes as well as the count of bone marrow T-cells are not associated with types of tumor cells. Conclusion. Arrest of hematopoiesis and increasing number of monocytes in blood correlate with the degree of bone marrow tumor infiltration and are not affected by morphoimmunological characteristics of FL tumor cells.

Pan-HDAC Inhibitors May Restore PRDM1 Expression in Follicular Lymphoma

Follicular lymphoma (FL) arises from a germinal center (GC) B cell proliferation supported by a bidirectional crosstalk with tumor microenvironment, in particular with follicular helper T cells (Tfh). Besides BCL2 translocation hallmark, tumor B cells progressively acquire loss-of-function alterations within chromatin organization factor genes. Notably, frequent inactivating mutations in CREBBP acetyltransferase are found in B-cell lymphoma and drive deep epigenetic and transcriptional profile modifications. Our transcriptional analysis on highly purified cell fractions revealed that, conversely to normal GC B cells (centroblasts and centrocytes), primary FL B cells are unable to upregulate the transcription repressor PRDM1 that is required for plasma cell differentiation and maintenance. This defect happens although FL tumor environment is enriched in the potent inducer of PRDM1, IL-21 that we found highly produced by CD4+ Tfhs.In 20 different FLs perfectly characterized at clinical and genetic level, we analyzed their FL cells obtained after purification from the initial tumor. Globally, we found a significant lack of IL-21-mediated PRDM1 response while our control cell line showed a positive increase of PRDM1 expression. We analyzed some of these primary FL tumor cells by qPCR-ChIP to assess BCL6 protein binding at intron 3 of the PRDM1 gene. We found that the lack of PRDM1 response to IL-21 was associated with an abnormal increased enrichment of the BCL6 protein repressor in PRDM1 gene. Our data indicated this was especially the case for FLs with nonfunctional CREBBP. We next explored 6 FLs, cells were treated in vitro with vorinostat, a pan-HDAC inhibitor. We found that for FL B cells carrying CREBBP loss-of-function mutations, vorinostat restored partially their PRDM1 response to IL-21 by lowering BCL6 bound to PRDM1. Finally, we wanted to assess this drug effect in the context of FL patients treated with a pan-HDACi. We found 4 patients involved in a phase I/II clinical at the Gustave Roussy Institute in Villejuif and treated with abexinostat, a new and off-label pan-HDAC inhibitor. All the patients presented a progressive FL disease, were heavily pretreated and their tumor were characterized by genomic DNA sequencing. One patient reached a stable complete remission, the drug was discontinued after 4 years of treatment. Among the three other patients, one had an objective 28-month response to the drug before relapse, one a stable disease for seven months while the last one did not response to the drug. For these 3 patients we've got fine needle tumor biopsy when patients re-progressed. Re-biopsies showed an increased expression of plasma cell-identity genes, mainly PRDM1 and XBP1, which underline the progression of FL B cells in the process of differentiation. Altogether, our data uncover a new mechanism by which pan-HDAC inhibitors may act positively to treat FL patients. DisclosuresRibrag:BMS: Consultancy, Honoraria, Other: travel; argenX: Research Funding; Servier: Consultancy, Honoraria; epizyme: Consultancy, Honoraria; NanoString Technologies: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Research Funding; Roche: Honoraria, Other: travel; Incyte Corporation: Consultancy; pharmamar: Other: travel; Infinity: Consultancy, Honoraria; MSD: Honoraria.

Regulatory T Cells Are Depleted in Low-Grade Lymphoma By the Combination of Local Low-Dose Radiation Followed By Intratumoral CpG-ODN

BACKGROUND: Non-Hodgkin B cell lymphomas are often infiltrated by immune effector cells including T, NK and dendritic cells. But despite their presence within the tumor they fail to control tumor growth. Some T cells can even play a supportive role to maintain the tumor and prevent the function of anti-tumor immune cells.Regulatory T cells (TRegs) function normally to dampen immune responses and prevent auto-immunity by direct cell contact or by secreting suppressive cytokines i.e. TGF-b and Interleukin-10. Follicular Lymphoma tumor cells can induce the conversion of CD4 T cells into TRegs (1-3), thereby evading anti-tumor immune responses.Follicular helper T cells (Tfh), another class of regulatory T cells, are found within normal follicles of lymphoid organs. Migrating into the germinal center, they promote the survival and differentiation of follicular B cells. In lymphoma, Tfh cells can send a survival signal to tumor B cells through CD40L/CD40 interactions (4).STUDY: In an ongoing multi-center phase I clinical trial (NCT02266147), patients with previously untreated low-grade lymphoma receive low dose (2Gyx2) radiotherapy to a single, tumor site, followed by intratumoral injection into the same site of a CpG-ODN (SD-101, Dynavax Technologies), an immune-modulatory molecule that targets Toll-like Receptor 9 (TLR-9). Doses ranged from 1mg to 8mg per injection in successive cohorts. A fine needle aspirate (FNA) of the treated site is performed before, one week after the first injection, and 1 week after the 5th and final weekly injection of CpG-ODN. FNA samples are sent to a central site by overnight delivery in media with 5% fetal calf serum. They are processed within 24 hours and stained for flow cytometry with panels of antibodies to delineate T, B, NK, dendritic, myeloid cells, and their subsets. Antibodies against activation antigens, as well as T cell exhaustion, inhibition and function are also used to characterize these cells.To date, 12 patients have been entered into the dose escalation phase of this study: 10 follicular lymphoma, 1 chronic lymphocytic lymphoma and 1 small lymphocytic lymphoma. Nine of 12 patients had FNAs that yielded enough viable cells for evaluation pre and 1 week post treatment.RESULTS: Therapy has been well tolerated. Local injection site reactions and fever, the most common side effects, resolved by 48 hours. There have been no related SAEs. The treated site regressed in all patients per CT scan at 3 months post treatment. The early evaluation of untreated lesions documented 1 PR. The remaining patients have stable disease with all but one having systemic tumor regressions ranging from 14% to 28%.Seven out of 9 patients evaluable by flow cytometry had an increase in total T cells at the treated site 1 week following the first dose of CpG ranging from 18 to > 300% above baseline in both the CD4 and CD8 compartments. More interestingly, there was a reduction of TRegs as a percentage of total T cells in 7 of 9 patients (average reduction: 21.3 ± 10.6%). A single patient with an increase in untreated tumor burden of 15% showed no reduction in TRegs. There was also a reduction of Tfh cells in 8 of the 9 evaluable patients (average reduction: 87.2 ±7.2%). One patient with SLL had no Tfh cells pretreatment. Figure 1A and B shows a representative example of TReg and Tfh cell depletion within the same FL patient.CONCLUSION: Immune modulating therapies can be delivered directly into a tumor to minimize systemic toxicity and induce changes in the tumor microenvironment while potentially inducing a global anti-tumor immune response. In this case, the combination of intratumoral CpG and low-dose radiation reduced the proportion of TRegs and Tfh cells, thereby modulating their immune inhibitory effects and tumor growth promoting effects, respectively. This clinical trial is ongoing and open to accrual.Monitoring the cell populations in the tumor site by repetitive sampling and analysis by flow cytometry reveals the pharmocodynamic effects of anti-cancer therapies, especially those intended to trigger anti-tumor immune responses. This knowledge can be used to tailor therapies and to influence the choice and scheduling of combinations of agents designed to target specific cell populations.1. Yang Z-Z, et al. Blood. 2007;110:537-44.2. Ai WZ, et al. Int J Cancer. 2009 Jan 1;124(1):239-44.3. Mittal S, et al. Blood. 2008;111:5359-70.4. AmŽ-Thomas P, et al. Blood. 2015 Apr 9;125(15):2381-5 [Display omitted] DisclosuresBartlett:Gilead: Consultancy, Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Millennium: Research Funding; Colgene: Research Funding; Medimmune: Research Funding; Kite: Research Funding; Insight: Research Funding; Seattle Genetics: Consultancy, Research Funding; MERC: Research Funding; Dynavax: Research Funding; Idera: Research Funding; Portola: Research Funding; Bristol Meyers Squibb: Research Funding; Infinity: Research Funding; LAM Theapeutics: Research Funding. Gordon:Northwestern University: Employment; Dr Leo I. Gordon: Patents & Royalties: Patent for gold nanoparticles pending. Coffman:Dynavax Technologies: Employment. Janssen:Dynavax Technologies: Employment. Levy:Bullet Biotechnology, Inc.: Consultancy.

Prosurvival and Chemoprotective Effects of Tumor-Associated Macrophages Reversed By Anti-CSF-1R Monoclonal Antibody in B-Cell Lymphomas

The results of gene expression profiling (GEP) and immunohistochemical studies indicate that survival is worsened by macrophages (MΦ) in the tumor microenvironment of various B-cell lymphomas including follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Tumor-associated macrophages (TAMs) are known to be different from other types of MΦ, but the effects of TAMs that worsen prognosis in B-cell lymphoma are essentially unknown, as are the mechanisms of these effects. Here, we determined the phenotype and effects of TAMs on tumor survival, proliferation, and drug resistance in B-cell lymphomas and evaluated strategies to reverse their effects.As compared to peripheral blood monocytes (Mo) from normal donors (ND), Mo from FL patients were differentiated less into M1 MΦ (defined as CD68+CD163loCD206loCD86hi) by culture with CSF-1 for 5 days followed by IFN-g + LPS for 2 days more. In contrast, Mo from FL patients and ND were differentiated similarly into M2 MΦ (defined as CD68+CD163hiCD206hiCD86lo) by culture with CSF-1 followed by IL-4. Consistent with this, MΦ gene signatures from FL tumors were more similar to previously-described signatures of M2 rather than M1 MΦ (Martinez et al, J Immunol, 2006, 177(10):7303-11).In co-culture, primary FL tumor cells and lymphoma cell lines (including RL, a transformed FL cell line; Granta 519, a mantle cell lymphoma (MCL) cell line; and Raji, a Burkitt lymphoma cell line) induced differentiation of Mo into MΦ. Differentiation could be prevented by CS4 monoclonal antibody (mAb), a fully human IgG1 anti-human CSF-1R mAb (ImClone/Eli Lilly), but not isotype control Ab. Elevated levels of CSF-1 in culture supernatants after addition of CS4 mAb and real-time PCR of tumor cells suggested secretion of CSF-1 by lymphoma cells. Spontaneous apoptosis of primary FL and MCL tumor cells, determined by Annexin V and propidium iodide staining, was significantly reduced by co-culture with ND Mo (p<0.01), whether pre-differentiated into MΦ with CSF-1 or not, but this protection could be reversed by CS4 mAb. Mo and/or pre-differentiated MΦ protected primary FL and MCL tumor cells from cytotoxic effects of doxorubicin and/or bendamustine (p<0.01), but CS4 mAb reversed this effect. To assess effects of MΦ on proliferation, lymphoma cell lines (RL, Granta 519, and Raji) were CFSE-labeled prior to co-culture with Mo and doxorubicin, and proliferation assessed by CFSE dilution by flow cytometry in the presence or absence of CS4 or isotype control mAbs. MΦ promoted proliferation of all three cell lines, but this effect could be reversed by CS4 mAb.To further understand the mechanism by which MΦ promote tumor survival and growth, we performed phosflow analysis and found increased phosphorylation of STAT3 in co-cultured lymphoma cells. Consistent with this, we observed a correlation between an 11-gene STAT3 activation signature, described by Huang et al in DLBCL tumors (J Clin Oncol, 2013, 52.8414), and a MΦ gene signature in whole genome GEP studies of 191 FL tumors (Pearson correlation co-efficient=0.396, p<0.001).In conclusion, our results suggest that Mo from FL patients are predisposed to differentiate into an M2-like MΦ state. The interaction between lymphoma cells and Mo/MΦ is reciprocal: a change in Mo (MΦ differentiation) induced by interaction with lymphoma tumor cells leads to a change in the tumor cells (promotion of survival, proliferation, and chemoresistance). More importantly, our results demonstrate that targeting TAMs using CS4, an anti-CSF-1R mAb, can be an effective strategy to overcome the adverse effects of TAMs and reverse chemoresistance. Further studies are needed to determine whether STAT3 activation contributes to the protumor effects of TAMs. This may provide novel insights into the molecular mechanisms related to TAMs and lymphoma cells and offers additional targets for therapeutic development. In the long term, strategies targeting TAMs is especially appealing, as they should be able to be combined with existing therapies including chemotherapy, other immunotherapy, and targeted therapy, potentially improving their efficacy without increasing toxicity for FL, DLBCL, and other B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Cross Talk between Follicular Th Cells and Tumor Cells in Human Follicular Lymphoma Promotes Immune Evasion in the Tumor Microenvironment

The microenvironment of human follicular lymphoma (FL), an incurable B cell non-Hodgkin's lymphoma, is thought to play a major role in its pathogenesis and course. Microenvironmental cells of likely importance include follicular Th cells (TFH) and regulatory T cells (Tregs), and understanding their interactions with FL tumor cells is necessary to develop novel therapeutic strategies. We found that IL-4 and CD40L are expressed by intratumoral TFH and induce production of CCL17 and CCL22 by FL tumor cells. IL-4 alone induces only CCL17 but enhances stimulation by CD40L of both CCL17 and CCL22. Consistent with our in vitro results, mRNA transcripts of IL-4 correlated with CCL17, but not CCL22, in gene expression profiling studies of FL biopsies, whereas CD40L correlated with both CCL17 and CCL22. Tumor supernatants induced preferential migration of Tregs and IL-4-producing T cells rather than IFN-γ-producing T cells, and Abs to CCR4 significantly abrogated the migration of Tregs. Our results suggest that through two distinct mechanisms, intratumoral TFH induce production of CCL17 and CCL22 by FL tumor cells and facilitate active recruitment of Tregs and IL-4-producing T cells, which, in turn, may stimulate more chemokine production in a feed-forward cycle. Thus, TFH appear to play a major role in generating an immunosuppressive tumor microenvironment that promotes immune escape and tumor survival and growth. Our results provide novel insights into the cross talk among TFH, tumor cells, and Tregs in FL, and offer potential targets for development of therapeutic strategies to overcome immune evasion.

High PD-1 expression and suppressed cytokine signaling distinguish T cells infiltrating follicular lymphoma tumors from peripheral T cells

AbstractDefects in T-cell function in patients with cancer might influence their capacity to mount efficient antitumor immune responses. Here, we identified highly reduced IL-4–, IL-10–, and IL-21–induced phosphorylation of STAT6 and STAT3 in tumor-infiltrating T cells (TILs) in follicular lymphoma (FL) tumors, contrasting other non-Hodgkin lymphoma TILs. By combining phospho-protein–specific flow cytometry with several T-cell markers, we identified that CD4+CD45RO+CD62L− FL TILs were largely nonresponsive to cytokines, in contrast to the corresponding autologous peripheral blood subset. We observed differential expression of the inhibitory receptor PD-1 in FL TILs and peripheral blood T cells. Furthermore, CD4+PD-1hi FL TILs, containing TFH and non-TFH cells, had lost their cytokine responsiveness, whereas PD-1− TILs had normal cytokine signaling. However, this phenomenon was not tumor specific, because tonsil T cells were similar to FL TILs. FL tumor cells were negative for PD-1 ligands, but PD-L1+ histiocytes were found within the T cell–rich zone of the neoplastic follicles. Disruption of the microenvironment and in vitro culture of FL TILs could restore cytokine signaling in the PD-1hi subset. Because FL TILs in vivo probably receive suppressive signals through PD-1, this provides a rationale for testing PD-1 Ab in combination with immunotherapy in patients with FL.

Cross-Talk Between Tumor B Cells, Follicular Dendritic Cells, and Follicular Helper T Cells: Implications for Follicular Lymphoma Pathogenesis and Therapy,

Abstract 3696 Background:The stromal microenvironment is thought to play a critical role in supporting the survival and growth of follicular lymphoma (FL) tumor cells. However, the key stromal factors involved are not well characterized. We hypothesized that FL tumor cells hijack the survival and growth mechanisms that are usually used by normal B cells in the lymph node microenvironment. For example, follicular dendritic cells (FDC) residing in germinal centers of lymph nodes play a vital role in supporting normal B cell survival and proliferation through adhesion molecules and by secreting chemokines and cytokines. In normal lymph nodes, lymphotoxin-alpha (LTA, TNFSF1) secreted by B cells activates FDC through LTbR and induces secretion of BAFF (TNFSF13B) and CXCL13. CXCL13 recruits follicular helper T cells (Tfh) into the B cell zone and CD40L (expressed by Tfh) together with BAFF prevents apoptosis and promotes proliferation of B cells. Here, we determined whether a similar cross-talk exists between FL tumor B cells, FDC, and Tfh. Methods:FL tumor samples were processed into single-cell suspension and cryopreserved in aliquots for later use. Primary tumor B cells were isolated by magnetic cell separation from single-cell suspensions of FL. The FDC-like cell line HK derived from human tonsil was used in place of primary FDC. Levels of BAFF, CXCL13, and LTA in culture supernatants were determined by ELISA. Apoptosis of tumor cells was determined by Annexin V assay and proliferation by tritiated thymidine incorporation assay. Activation of NFkB was determined by the TransAM NFkB p52 ELISA kit. Results:We developed a co-culture model to mimic the FL tumor microenvironment to study the interactions between tumor cells, FDC and Tfh. We observed that FL tumor cells spontaneously produced low levels of LTA but its production was significantly enhanced when tumor cells were cultured with either recombinant BAFF or CD40L. Dose response curves from FL tumor cells treated with both BAFF and CD40L recombinant proteins showed synergistic increase in LTA secretion. Recombinant BAFF and CD40L also synergistically enhanced the survival and proliferation of FL tumor cells. HK cells spontaneously produced low levels of BAFF and CXCL13 but their production was significantly enhanced by recombinant LTA in a dose-dependent manner.Consistent with the above studies, co-culturing FL tumor cells with HK cells significantly enhanced LTA secretion, possibly due to the effects of BAFF secreted by HK cells. In addition, in this co-culture, we also observed enhanced production of BAFF and CXCL13 possibly due to the effects of LTA secreted by the tumor cells. The production of BAFF and CXCL13 in this co-culture could be inhibited by an LTA neutralizing antibody in a dose-dependent manner suggesting the role of LTA in this mechanism. Co-culturing FL tumor cells with HK cells also significantly enhanced the survival and proliferation of FL tumor cells greater than two-fold compared with FL tumor cells alone. This effect also could be inhibited by LTA neutralizing antibody but not isotype control antibody. The survival benefit for tumor cells was further enhanced upon co-culture with autologous Tfh cells FACSorted from the same tumor sample. We also studied the downstream NFkB signaling mediated by BAFF on tumor B cells and by LTA on HK cells in the co-culture. We found that both tumor B cells and HK cells had significantly higher NFkB activity upon co-culture compared to cells alone. Conclusions:Our results suggest a model where LTA secreted by tumor B cells forms the afferent axis and induces production of BAFF and CXCL13 by FDC. CXCL13 preferentially recruits CXCR5-expressing Tfh into the tumor microenvironment and CD40L expressed by Tfh and BAFF secreted by FDC form the efferent axis and promote further production of LTA by tumor cells. In addition, CD40L and BAFF synergistically enhance the survival and proliferation of FL tumor cells via activation of NFkB. Our results suggest that because of their redundant functions, blocking BAFF-BAFF-R pathway alone or CD40-CD40L pathway alone with monoclonal antibodies as is being done in ongoing clinical trials may not be optimal. In contrast, blocking both pathways simultaneously is likely to significantly enhance the clinical responses. Our results also suggest that neutralizing LTA or blocking its receptor may be another novel therapeutic strategy for management of FL. Disclosures:No relevant conflicts of interest to declare.

PD-1+ T Cell Subsets in Follicular Lymphoma Tumor Microenvironment and Their Implications for Prognosis and Therapy

Abstract 2648 Background:Programmed death (PD)-1, a coinhibitory receptor expressed by effector T cells (Teffs) is highly expressed on intratumoral T cells (mean 61%, range 34–86% for CD4+ T cells and mean 44%, range 31–69% for CD8+ T cells) in follicular lymphoma (FL), a finding associated with impaired ability to recognize autologous tumor (Nattamai et al, ASH 2007). Hence, PD-1 expression would be expected to confer an unfavorable prognosis in FL. However, correlation of PD-1 with clinical outcome in FL has been inconsistent with two studies showing favorable (Carreras et al, J Clin Oncol 2009; Wahlin et al, Clin Cancer Res 2010) and one study showing unfavorable (Richendollar et al, Hum Pathol 2011) outcome. While differences in method of analysis and type of treatment may explain the disparate results, a more complex model may be necessary to understand the prognostic impact of PD-1 in FL as PD-1 is expressed not only on antitumor Teffs but also on protumor follicular helper T cells (Tfh) and regulatory T cells (Tregs). Methods:To determine the nature of PD-1+ T cells in FL we performed comprehensive genomic and immunologic studies. By flow cytometry, we observed that the intratumoral CD4+ T cells in FL may be categorized into 3 subsets based on PD-1 expression - PD-1 high (PD-1hi), intermediate (PD-1int), and low (PD-1lo). The intratumoral CD8+ T cells consisted of PD-1int and PD-1lo subsets. The 3 CD4+ T cell subsets were FACSorted from FL tumors (n=3) and whole genome gene expression profiling (GEP) was performed. T cell subsets sorted similarly from tonsils served as controls for reactive follicular hyperplasia (FH) (n=3). Differentially expressed genes in GEP studies were confirmed at the mRNA level by real-time PCR (n=5) and at the protein level by flow cytometry when antibodies were available (n=5–10). Results:Our results suggested that CD4+PD-1hi T cells are Tfh cells (CXCR5hiBcl6hi ICOShiCD40LhiSAPhiPRDM1loIL-4hiIL-21hi); the CD4+PD-1int T cells consisted of a mixture of activated Teffs (CD45RO+CD45RA−) including Th1 (Tbet+IFNg+), Th2 (IL-10+), and Th17 cells (RORc+IL-17+), and Tregs (Foxp3+CD25hiCD127lo); and the CD4+PD-1lo T cells consisted of a mixture of activated Teffs (CD45RO+CD45RA− but IFNg−IL-4−IL-10−IL-17−), Tregs, and naïve T cells (CD45RO−CD45RA+CCR7+). Although these subsets were present in both FL and FH, there were important differences. IL-4 expression was significantly higher in Tfh in FL vs. FH and may play a role in the pathogenesis of FL. IL-17 expression was low and expression of coinhibitory molecules BTLA and CD200 was high in CD4+PD-1int T cells in FL vs. FH. BTLA and CD200 were also increased in CD8+PD-1int T cells in FL vs. FH. However, other coinhibitory molecules (LAG-3, Tim-3, CD160, CTLA-4, CD244, KLRG1) were not significantly different between FL and FH. CD4+PD-1int T cells also had higher expression of BATF, a transcription factor associated with T cell exhaustion in FL vs. FH. Together, these results suggest that the CD4+PD-1int T cells in FL may be in a state of T cell exhaustion whereas the CD4+PD-1int T cells in FH may represent recently activated Teffs. Consistent with this, blocking PD-1 with anti-PD-1 blocking antibody significantly enhanced proliferation and the production of Th1 (IFNg, TNFa) but not Th2 (IL-4, IL-5, IL-10, IL-13) cytokines by intratumoral CD4+ and CD8+ T cells in response to stimulation with autologous FL tumor cells (n=3). As expected, Tregs were increased in number in FL vs. FH and were present in the PD-1int and PD-1lo T cell subsets. We found 74% (range 40–97%) of FL Tregs expressed PD-1. Among the CD4+PD-1lo and CD8+PD-1lo T cells, there were more activated Teffs and fewer naïve T cells in FL vs. FH. Conclusions:Our results suggest that the PD-1+ T cells in FL are comprised of a mixture of antitumor Teffs and protumor Tfh and Tregs. The prognostic impact of PD-1+ T cells in FL may dependent on the relative frequency of these subsets as ligation of PD-1 may produce favorable (inhibition of protumor Tfh and Tregs) or unfavorable (inhibition of antitumor Teffs) outcomes by inhibiting or promoting tumor growth, respectively. Conversely, our results imply that agents that block PD-1/PD-ligand pathway may have the opposite effect on these T cell subsets and enumeration of the intratumoral PD-1+ T cell subsets may serve as biomarker to predict response to these agents in FL and possibly other B-cell malignancies. Disclosures:Dong:GSK: Consultancy; Genentech: Honoraria; Tempero: Consultancy; Ono: Consultancy; AnaptysBio: Consultancy. Neelapu:Cure Tech Ltd: Research Funding.

Role of IL-4 in Inducing Immunosuppressive Tumor Microenvironment in Follicular Lymphoma

Abstract 771 Background:Recently, using proteomic profiling of tumor lysates, Calvo et al (Blood, 2008) found that IL-4 levels were significantly higher in follicular lymphoma (FL) tissues than in tissues from follicular hyperplasia. IL-4 present in the tumor microenvironment may promote the survival and growth of tumor B cells and may play a role in the pathogenesis of FL. However, the source of IL-4 production was not evaluated in these studies. Several reports have shown that regulatory T cells (Tregs) that can inhibit the function of antitumor CD4+ and CD8+ T cells are increased in number in the tumor microenvironment in FL. Tregs can be induced from conventional CD4+ T cells by the tumor cells but may also be actively recruited to the tumor site by CCL17 and CCL22 produced by tumor cells. However, B cells are not known to constitutively express either of these chemokines. As IL-4 is increased in the FL tumor microenvironment and can activate STAT6, and CCL17 and CCL22 are STAT6-responsive chemokine genes, we tested the possibility that these are interrelated. Methods:Surgical tumor samples from FL patients were processed into a single-cell suspension and cryopreserved for later use. Tumor cells isolated by magnetic cell separation were cultured in the presence or absence of IL-4 and chemokine levels were assessed in the supernatants after 48 hours by ELISA. Intracellular cytokine assay was performed on intratumoral T cells and tumor cells to determine the source of IL-4. Western blotting and phosflow was performed to assess the phosphorylation status of STAT6. Migration of Tregs in response to tumor supernatants was assessed in a transwell chemotaxis assay. Results:Using primary human FL samples we demonstrate that IL-4 is secreted by intratumoral follicular helper T cells (Tfh) and more importantly, IL-4-producing CD4+ T cells were increased in number in the FL tumor microenvironment as compared with benign follicular hyperplasia. While FL tumor cells produced CCL22 constitutively, the production of CCL17 was induced by IL-4 via activation of STAT6. Consistent with this, we observed a direct correlation between IL-4 and CCL17 mRNA transcripts in whole genome microarray studies of FL tissues (r = 0.41, P = <0.0001). The expression of CCL17 by tumor B-cells was dependent on STAT6 since knockdown of STAT6 using siRNA inhibited its production. IL-4 also stimulated CCL17 secretion from tumor cells from other B-cell lymphomas such as diffuse large cell lymphoma, mantle cell lymphoma, and splenic marginal zone lymphoma as well as intratumoral T cells in FL. CCL17 produced by these cells induced preferential migration of Tregs and IL-4–producing T cells than IFN-g–producing T cells. Antibodies to CCL17 and CCR4 significantly abrogated the migration of Tregs in response to tumor supernatants. Conclusions:Our results suggest that IL-4 produced by Tfh cells in FL induces CCL17 production through autocrine and paracrine effects on intratumoral T cells and tumor cells, respectively, and facilitates active recruitment of regulatory T cells and IL-4–producing T cells that in turn may stimulate more CCL17 production. Thus, IL-4 creates a vicious cycle and leads to an immunosuppressive tumor microenvironment that promotes immune evasion and tumor survival and growth. Our results provide novel insights into the role of IL-4 in immunosuppression in FL and other B-cell lymphomas and offer potential targets for development of therapeutic strategies to overcome immune evasion in these malignancies. Characters:2,993 (3,800 max without spaces) Disclosures:No relevant conflicts of interest to declare.

Improving the treatment outcome in follicular lymphoma using rituximab maintenance

Follicular lymphoma (FL) is the second most frequent lymphoma, representing approximately 70% of all indolent lymphomas and approximately 20% of all nonHodgkin’s lymphomas (NHLs) in adults [1]. Patients with FL usually have a long overall survival (OS) time, but disease progression typically occurs 3–5 years after initiation of treatment. There is no commonly accepted standard frontline therapy for FL patients. For several years a broad range of therapeutic options were available; however, historical studies did not show a survival benefit of one particular regimen. The real progress in FL treatment has been made after the introduction of immunochemotherapy with rituximab, an anti-CD20 monoclonal antibody. The CD20 antigen is expressed on the surface of both normal and NHL B-cells, although it is in especially high density on FL tumor cells [2]. The addition of rituximab to standard polychemotherapy regimens such as cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) or cyclophosphamide, vincristine and prednisone (CVP), resulted in a significant increase in overall response (OR), complete response (CR) and time-to-progression (TTP) rates in FL [3–6]. In a study conducted by Hiddemann et al., 428 patients with untreated, advanced-stage FL were randomly assigned to therapy with CHOP alone or CHOP combined with rituximab (R-CHOP) [7]. R-CHOP reduced the relative risk for treatment failure by 60% and significantly prolonged TTP (p < 0.001). In addition, patients treated with R-CHOP had a significantly higher OR rate (96 vs 90%; p = 0.011) and prolonged time of remission (p = 0.001), as well as superior OS (p = 0.016). In a large, randomized, multicenter study, the combination of rituximab with a CVP regimen (R-CVP) resulted in a significant increase in both OR and CR rates compared with patients treated with CVP alone (81 and 41 vs 57 and 10%, respectively; p < 0.0001) [8]. According to the current recommendations, immunochemotherapy with rituximab used in combination with CVP or CHOP regimens, but also with purine nucleoside analog-based schemes or bendamustine, should be applied in FL patients with progressive, symptomatic disease [9]. However, despite distinct progress in FL treatment, the disease still remains incurable. Different maintenance regimens were assessed to provide means for improving progression-free survival (PFS) and OS of indolent NHL patients. Numerous randomized trials examined the benefit of IFN-a consolidation or maintenance therapies for indolent NHL, including FL [10,11]. In the Southwest Oncology Group (SWOG) study, 279 indolent lymphoma patients in advanced clinical stage III and IV, who responded to the induction treatment with prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, mechlorethamine, vincristine and procarbazine (ProMACE-MOPP), were randomly assigned either to consolidation with IFN-a or to the observation “...maintenance treatment with rituximab should be considered for all [follicular lymphoma] patients responding to first-line therapy, as a new standard of care in previously untreated [follicular lymphoma] patients.”

Clinical Translation of a Prognostic Follicular Lymphoma Signaling Profile

AbstractAbstract 636 Introduction:Phospho-flow cytometry signaling profiles have great potential to identify and monitor negative prognostic subpopulations of cells within tumors. We recently reported that altered B cell receptor (BCR) signaling identifies a subset of lymphoma negative prognostic (LNP) cells within follicular lymphoma (FL) tumors from patients with shorter overall survival (Irish et al., PNAS 2010). Each 1% increase in LNP cells observed within the tumor at diagnosis increased the patient's annual risk of death by 2.5%. However, before signaling profiles such as this can be used in the clinic, the assay must be simplified and translated from the complex and time-consuming version used in research studies. Here we report a prognostic signaling profile suitable for follicular lymphoma clinical specimens of less than one million cells. Methods:To simplify LNP cell counting in FL patient specimens, detection of six key phospho-protein readouts for BCR signaling was combined on one cytometer channel, creating a single readout for BCR network signaling activity (termed p-BCR network). This combined stain measured phosphorylation of SYK, PLCγ, BLNK, Src family kinases (SFK), STAT5, and ERK using Alexa 488 dye conjugates of each phospho-specific antibody. LNP cells are defined by a lack of BCR signaling response at all of these phospho-protein readouts.Upon receipt of a patient's tumor sample, 0.25 million cells were stimulated for 4 minutes by polyclonal F(ab')2 against IgM and IgG (10 μ g/mL each; α-BCR), left unstimulated as a negative control, or exposed to a positive control stimulus (α-BCR + H2O2). Samples were stained with a four-color flow cytometry panel detecting p-BCR network, lymphoma B cell markers BCL2 and CD20, and CD3 or CD5 as a marker for T cells. Approximately 50,000 lymphoma B cells were collected and LNP cells quantified as before. The p-BCR network assay typically required 2h to complete. In parallel, LNP cells were quantified in samples from the same specimens using the original method, where phospho-proteins were detected individually using a set of staining panels. An additional control staining panel measured total tyrosine phosphorylation (p-Y) in place of p-BCR network staining in the four-color panel. Results:LNP cell counts measured by combined p-BCR network stain using 0.75 million FL tumor cells total were equivalent to those obtained by individual measurement of phospho-proteins (R2 = 0.98). Critically, in the combined p-BCR network stain the same patterns of signaling in lymphoma cell subpopulations were observed as when individual phospho-proteins were measured. Thus, not only was the same quantity of LNP cells detected by the p-BCR network stain, the same subpopulations of cells were identified by the two techniques. Total phospho-tyrosine following BCR stimulation was examined as a potential surrogate for p-BCR network staining, but high basal p-Y signaling and additional BCR mediated p-Y signaling obscured the LNP cell subset in some patient specimens. Thus, a total p-Y readout could not be used in place of the p-BCR network readout or detection of individual phospho-protein readouts. Conclusions:A clinical prognostic follicular lymphoma signaling profile can be measured in small specimens of live tumor cells, such as fine needle aspirates, and collected using commonly available flow cytometers. These results create new opportunities to use signaling profiles to identify FL patients who might benefit from clinical trials and to monitor emergence of negative prognostic lymphoma cells over time following therapy. Disclosures:No relevant conflicts of interest to declare.

Phase II safety and efficacy study of CT-011, a humanized anti-PD-1 monoclonal antibody, in combination with rituximab in patients with relapsed follicular lymphoma.

TPS305 Background: Follicular lymphoma (FL) is one of the most immune-responsive of all cancers. However, immunosuppressive mechanisms in the tumor microenvironment may impair the efficacy of antitumor immune responses. We have recently identified the programmed death (PD)- 1/PD-ligand pathway as an important immunosuppressive mechanism in the FL tumor microenvironment. Expression of the coinhibitory PD-1 receptor was markedly increased on intratumoral T cells and associated with decreased reactivity to autologous FL tumor cells. Blocking PD-1/PD-ligand pathway with CT-011 enhanced the proliferation of antitumor T cells and augmented the expression of activating receptors on human NK cells. Administration of CT-011 delayed tumor growth and improved survival of mice in lymphoma xenograft models and combination with rituximab further improved survival compared with rituximab alone (Chu et al, Blood, Nov 2009; 114: 724.). In a phase I clinical trial, CT-011 was safe, induced sustained elevations of CD4+ T cells, and the sole patient with FL on the trial, who had large tumor masses, achieved a durable complete remission lasting >14 months (Berger et al, Clin Cancer Res, 2008). Based on these data, we are now conducting a single-arm phase II trial with CT-011 and rituximab in relapsed FL patients (n = 30). The rationale is that the combination is likely to activate both innate (NK cells) and adaptive (T cells) arms of the immune system and thereby enhance the clinical efficacy without increasing toxicity. The primary endpoint is overall response rate. Methods: Patients with FL, relapsing after at least 1 but not more than 4 prior therapies, with rituximab-sensitive disease will be eligible. Patients must be ≥ 18 years, have measurable disease, adequate organ function, and absolute lymphocyte count of ≥ 0.6×109/L. Patients with HIV, hepatitis B or C, and autoimmune diseases are excluded. CT-011 will be administered IV every 4 weeks for 4 infusions and rituximab will be administered IV weekly for 4 weeks. The trial was activated in January 2010 and one patient has been enrolled. Blood and tumor samples will be collected to assess the effect of CT-011 on T cells and NK cells. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration CureTech

Lenalidomide Repairs Suppressed T Cell Immunological Synapse Formation in Follicular Lymphoma

Identifying mechanisms of tumor-induced immune suppression will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired T cell responses in follicular lymphoma (FL) and demonstrate impaired T cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between tumor-infiltrating CD4 and CD8 T cells and autologous FL tumor cells with and without superantigen as antigen-presenting cells (APCs). We identified a significant reduction in formation of the T cell immune synapse in both CD4 (p<0.01) and CD8 T cells (p<0.01) from FL patients compared to age-matched healthy donors (> 50% reduction). Comparable immunological defects were also identified in CD4 and CD8 T cells from transformed-FL (t-FL) patients. This defect was induced by tumor contact since T cell defects were induced in healthy T cells when they were were co-cultured for 48 hr with either allogeneic FL or t-FL cells, but not with healthy allogeneic B cells (P < 0.01). Of interest there was enhanced suppression of CD4 T cell synapses following FL co-culture assays and CD8 T cells in the t-FL setting. Since previous gene expression profile studies (Dave et al. NEJM, 2003) demonstrated prognostic significance of altered expression of immune signature genes, we examined the molecular nature of the FL-induced T cell defect by quantifying recruitment of a number of these T cell cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with FL cells, previously healthy T cells showed suppressed recruitment of integrin LFA-1, Lck, Itk, Rab27A and filamin-A to the synapse in subsequent T cell:APC interactions (P < 0.01). We further demonstrate that the immune synapse defects were repaired in part by treatment of the cells with the immunomodulatory drug lenalidomide. Of note, treatment of both FL B cells and autologous T cells with lenalidomide (1 μM for 24h) was required to enhance formation of the F-actin synapse and recruitment of tyrosine-phosphorylated protein and filamin-A irrespective of the presence of exogenous antigen (P < 0.01). We validated the altered expression of a number of these molecules including Itk, Rab27A and filamin-A at the protein level in FL tissue microarrays (TMA). Of note, elevated expression of intrafollicular Rab27A, that mediates targeted secretion of CD8 T cell cytolytic granules, was found in samples from a long-survival group (FL patients who lived more than 15 years). These results provide mechanistic insight into the demonstrated activity of lenalidomide in relapsed or refractory aggressive FL (Wiernik et al. JCO, 2008). These studies demonstrate the role of the tumor in the observed T cell defects in FL, and the molecular basis of these defects and suggest that immunotherapeutic approaches including the use lenalidomide offer the exciting prospect of repairing T cell suppression in B cell malignancies to enhance immune-mediated immunological responses against lymphoma cells.