Follicular Lymphoma Microenvironment Research Articles

Integrative Analysis of Cell Crosstalk within Follicular Lymphoma Cell Niche: Towards a Definition of the FL Supportive Synapse.

Simple SummaryFollicular lymphoma, the most frequent indolent non-Hodgkin’s B cell lymphoma, arises from a germinal center B cell proliferation supported by a multidirectional crosstalk with the tumor microenvironment, in particular with follicular helper T cells and mesenchymal stromal cells. Here, we explored this complex network, starting from a comparative analysis of the molecular signatures of B cells, T cells, and stromal cells obtained from normal versus lymphoma tissues, and focusing on deregulated genes reflecting the crosstalk between these three cell subsets organizing the lymphoma cell niche. This helps us to point out new lymphoma-specific pathways, related to transcriptomic and functional specific features of T and stromal cells, and contributing to tumor B cell support directly or through the recruitment and/or activation of other pro-tumoral cell components. In the future, targeting these cell interactions with specific drugs in the FL niche could represent an attractive option for novel therapeutic strategies.Follicular lymphoma (FL), the most frequent indolent non-Hodgkin’s B cell lymphoma, is considered as a prototypical centrocyte-derived lymphoma, dependent on a specific microenvironment mimicking the normal germinal center (GC). In agreement, several FL genetic alterations affect the crosstalk between malignant B cells and surrounding cells, including stromal cells and follicular helper T cells (Tfh). In our study, we sought to deconvolute this complex FL supportive synapse by comparing the transcriptomic profiles of GC B cells, Tfh, and stromal cells, isolated from normal versus FL tissues, in order to identify tumor-specific pathways. In particular, we highlighted a high expression of IL-6 and IL-7 in FL B cells that could favor the activation of FL Tfh overexpressing IFNG, able in turn to stimulate FL B cells without triggering MHC (major histocompatibility) class II expression. Moreover, the glycoprotein clusterin was found up-regulated in FL stromal cells and could promote FL B cell adhesion. Finally, besides its expression on Tfh, CD200 was found overexpressed on tumor B cells and could contribute to the induction of the immunosuppressive enzyme indoleamine-2,3 dioxygenase by CD200R-expressing dendritic cells. Altogether our findings led us to outline the contribution of major signals provided by the FL microenvironment and their interactions with malignant FL B cells.

PD1 distribution pattern, regardless of the cell origin, is an independent microenvironmental prognostic factor for progression-free survival in follicular lymphoma

Follicular lymphoma (FL) is a well-studied microenvironment-dependent hematological malignancy, but the crosstalk between various involved cell subtypes is still not fully understood. Recent promising results of immunotherapy in recurrent FL warrant the need for an in-depth analysis of the expression and role of immune system-related proteins in the FL microenvironment.Seventy-one patients with FL and available diagnostic paraffin blocks were enrolled in the retrospective analysis. Histopathological diagnoses were revised according to the World Health Organization recommendations. Patients were either observed (watch and wait/W&W group) or immediately treated with chemo(immuno)therapy regimens according to their clinical status. Immunohistochemical assessment of PD1, PDL1, CD4, CD8, CD163, CD68-KP1, CD68-PGM1 was performed. The scoring methods included both semi-quantitative estimation of positive cells and architectural pattern distribution. The differences between PD1 staining distribution and intensity were classified as intra/perifollicular vs. interfollicular/diffuse cells and presented bright vs. dim immunoreactivity, respectively. No statistically significant differences in the density distribution of the immunohistochemical stainings were found between W&W and chemo(immuno)therapy groups. Interfollicular/diffuse pattern of PD1 expression had significantly decreased progression-free survival when analyzing the whole cohort and patients on chemo(immuno)therapy (p = 0.014 and p = 0.07, respectively). The high dependence was not significant in the W&W group. PD1 positivity of cells did not correlate with CD4 or CD8 immunophenotype. Morphologically FL neoplastic cells were entirely PDL1 negative, but granular and membranous staining was detected in the FL microenvironment.In line with previous studies, PD1/PDL1 expression was predominantly localized in the FL microenvironment, indicating that FL cells might not be the direct target for anti-PDL1 therapy. However, we show that the localization of PD1 expression could be a viable progression-free survival biomarker for FL.

Prognostic value of tissue-infiltrating immune cells in tumor microenvironment of follicular lymphoma: A meta-analysis

BackgroundThe follicular lymphoma (FL) microenvironment is composed of follicular dendritic cells (FDCs), tumor-infiltrating CD4/CD8+ T cells (TILs), follicular regulatory T (Treg) cells, lymphoma-associated macrophages (LAMs), and immune checkpoint–related immune cells, all of which are relevant in the prognosis of FL, but their results remain controversial. Therefore, we performed this systematic review to explore the relationship between the FL microenvironment and prognosis. MethodsRelevant studies were identified from PubMed, EMBASE and the Cochrane Library. Twenty-three trials involving 3336 patients with FL were included for analysis. ResultsThis meta-analysis confirmed the unfavorable prognostic role of high CD21+/CD23+ FDC density in overall survival (OS) and progression-free survival (PFS). CD8+ or granzyme B+ TILs instead of CD4+ TILs are indicators for good OS. FoxP3+ Treg cells was not associated with prognosis, and even in subgroup analysis neither the number of cells nor the infiltration pattern had predictive value. A high degree of CD68+ macrophage infiltration was a negative prognostic factor for OS, but was associated with good prognosis in the rituximab-era subgroup. Although there was no correlation between PD1-positive immune cells and prognosis, subtypes with the follicular helper T (TFH) or exhausted T cell (TEX) phenotype tended to influence prognosis. The HR in the short time to transformation (TTT) analyses suggested that high CD68+ LAM numbers, diffuse pattern of FOXP3+ Treg cells and PD1+ cells, and high PD-L1 cell numbers are adverse factors leading to early transformation. ConclusionsMultiple tissue-infiltratingimmune cells in microenvironment play critical and different roles in FL prognosis.

Cathepsin S Alterations Induce a Tumor-Promoting Immune Microenvironment in Follicular Lymphoma

Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4+ Tcells invitro. Tumors with hyperactive CTSS showed increased CD4+ Tcell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment.

Aberrant Cathepsin S Induces a Supportive Immune Microenvironment in Follicular Lymphoma

The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. Here, we provide biochemical, structural, functional and clinical evidence that aberrant Cathepsin S (CTSS) activity induces a supportive immune microenvironment in FL. By targeted DNA sequencing of 305 diagnostic FL biopsies, we identified somatic mutations of CTSS in 8% of cases (24/305), mostly clustered at Y132 (19/24) converting Y to D (16/19). A subset of CTSS Y132 mutations (N=5) occurred at lower variant allele frequencies (5-10%), indicating subclonality. Another 13% of FL had CTSS amplifications (37/286). CTSS Y132 mutations and CTSS amplifications were mutually exclusive. In a cohort of 51 FL, CTSS amplifications were associated with higher CTSS expression (P=0.05). Of note, a subset of FL without CTSS amplifications also had higher CTSS expression, suggesting additional mechanisms of transcriptional dysregulation. CTSS is a cysteine protease that is highly expressed in endolysosomes of antigen presenting cells and malignant B-cells. CTSS is involved in proteolytical processing of antigenic peptides for presentation on MHC-II to be recognized by antigen specific CD4+ T-cells. CTSS is synthesized as an inactive zymogen, which is converted to its active form by autocatalytic cleavage of the autoinhibitory propeptide (pro-CTSS). We used CRISPR/Cas9 to introduce CTSS Y132D into Karpas422, a B-cell lymphoma cell line that harbors the FL hallmark translocation t(14;18). Single-cell derived Y132D mutant clones showed >3-fold higher ratios of active CTSS to pro-CTSS (N=4, P=0.0003). Immunoprecipitated CTSS Y132D had >3-fold higher in vitro substrate cleavage activity compared to CTSS wild type (WT) (N=6, P=0.001). We purified pro-CTSS WT and Y132D and assayed their in vitro autocatalytic cleavage over time. The time required to convert 50% of pro-CTSS decreased from 17 minutes for WT to 11 minutes for Y132D (N=3, P=0.04). In contrast, purified active CTSS WT and Y132 had similar in vitro cleavage activities. Molecular dynamics simulations showed that the Y132D mutation shortens the distances by ~2Å between the catalytic triad of active CTSS (C139, H278, N298) and a stretch of amino acids from the proform (L80, G81, D82, S94), which may facilitate intramolecular cleavage. By mass spectrometry we could indeed detect novel intermediate-sized CTSS fragments. Thus, Y132D does not increase the activity of the mature enzyme but is a gain-of-function mutation by accelerating the conversion from pro-CTSS to catalytically active CTSS. CD74 (invariant chain) is a physiologic CTSS substrate that plays critical roles in the assembly, trafficking and stabilization of peptide-free MHC-II. CTSS cleaves CD74, thereby allowing binding and presentation of antigens on MHC-II to antigen specific CD4+ T-cells. We could show that CTSS Y132D enhanced CD74 cleavage in Karpas422 cells. We then tested the impact of CTSS on antigen specific CD4+ T-cell activation in co-culture assays. CTSS knock-out lymphoma cells were broadly incapable of activating CD4+ T-cells. Overexpression of CTSS WT activated CD4+ T-cells more efficiently compared to empty vector control. CTSS Y132 had the highest capacity to stimulate antigen specific CD4+ T-cell responses. Furthermore, in primary FL biopsies (N=51) CTSS Y132 mutations had gene expression profiles linked with antigen-processing and chemokine perturbation, including CXCL13, a B-cell chemoattractant produced by activated CD4+ T-follicular helper cells. Lastly, we aimed to correlate CTSS aberrations with clinical outcome in patients who received standard immunochemotherapy (R-CHOP) for advanced FL (N=51 with available CTSS mutation and gene expression data). Compared to all other patients (N=34), patients with CTSS Y132 mutations or CTSS overexpression (N=17) had longer failure free survival (P=0.012) and overall survival (P=0.041). In summary, we propose that aberrant CTSS activity - even if only present in a FL subclone - can elicit a CD4+ T-cell driven tumor-promoting immune response, which could be amplified within the microenvironment via pro-inflammatory and chemotactic cytokines and substantially impact the biology and clinical course of the disease. Thus, aberrant CTSS activity is a promising biomarker and therapeutic target in FL and potentially also other tumors. Disclosures Klapper: Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Hiddemann:Bayer: Research Funding; Roche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Vector Therapeutics: Consultancy, Honoraria. Steidl:Juno Therapeutics: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Tioma: Research Funding; Seattle Genetics: Consultancy; Bayer: Consultancy. Kridel:Gilead Sciences: Research Funding. Weinstock:Celgene: Research Funding; Verastem Oncology: Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.

Pan-HDAC Inhibitors Restore PRDM1 Response to IL21 in CREBBP-Mutated Follicular Lymphoma.

Follicular lymphoma arises from a germinal center B-cell proliferation supported by a bidirectional crosstalk with tumor microenvironment, in particular with follicular helper T cells (Tfh). We explored the relation that exists between the differentiation arrest of follicular lymphoma cells and loss-of-function of CREBBP acetyltransferase.Experimental Design: The study used human primary cells obtained from either follicular lymphoma tumors characterized for somatic mutations, or inflamed tonsils for normal germinal center B cells. Transcriptome and functional analyses were done to decipher the B- and T-cell crosstalk. Responses were assessed by flow cytometry and molecular biology including ChIP-qPCR approaches. Conversely to normal B cells, follicular lymphoma cells are unable to upregulate the transcription repressor, PRDM1, required for plasma cell differentiation. This defect occurs although the follicular lymphoma microenvironment is enriched in the potent inducer of PRDM1 and IL21, highly produced by Tfhs. In follicular lymphoma carrying CREBBP loss-of-function mutations, we found a lack of IL21-mediated PRDM1 response associated with an abnormal increased enrichment of the BCL6 protein repressor in PRDM1 gene. Moreover, in these follicular lymphoma cells, pan-HDAC inhibitor, vorinostat, restored their PRDM1 response to IL21 by lowering BCL6 bound to PRDM1. This finding was reinforced by our exploration of patients with follicular lymphoma treated with another pan-HDAC inhibitor. Patients showed an increase of plasma cell identity genes, mainly PRDM1 and XBP1, which underline the progression of follicular lymphoma B cells in the differentiation process. Our data uncover a new mechanism by which pan-HDAC inhibitors may act positively to treat patients with follicular lymphoma through the induction of the expression of plasma cell genes.

PARP14 Is a Novel Therapeutic Target in STAT6 mutant Follicular Lymphoma

The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. The underlying molecular mechanisms and therapeutic vulnerabilities are not well understood.IL-4 producing follicular helper T cells (TFH) have been identified as a key component of the malignant B-cell niche. IL-4 activates paracrine signaling via STAT6. In a cohort of 258 patients with advanced stage FL, we identified STAT6 mutations in 13% of diagnostic biopsies (n=33). All mutations were clustered within the DNA binding domain, mostly at D419, and included a polymorphic variant (rs11172102). Gene set enrichment analysis (GSEA) revealed that STAT6 mutant cases were significantly enriched for two distinct IL-4 gene expression signatures. Gene expression data and immunohistochemistry of primary FL samples showed significant up-regulation of IL-4/STAT6 target genes in STAT6 mutant cases, including FCER2, which encodes for CD23.We stably expressed wild type STAT6 or mutant STAT6 (D419G, N421K, and D519V) in two B-cell lymphoma lines (OCI-Ly1, OCI-Ly8), both harboring the FL hallmark translocation t(14;18). Upon IL-4 stimulation, cells expressing mutant STAT6 had significantly increased FCER2 transcript levels. Similarly, IL-4 induced expression of membrane-bound as well as soluble CD23 was significantly increased in STAT6 mutant cells. Cells expressing mutant STAT6 showed significantly increased nuclear accumulation of pSTAT6 following IL-4 stimulation. Of note, we did not observe any effect of STAT6 mutations in the absence of IL-4.RNA sequencing of IL-4-stimulated lymphoma cell lines (STAT6 mutant versus wild type) identified PARP14 -a known transcriptional co-activator of STAT6- among the top differentially expressed genes. Bioinformatics and functional experiments demonstrated that PARP14 per se is a novel STAT6 target gene. Furthermore, reporter assays showed increased transactivation activity of mutant STAT6 at the PARP14 promotor, suggesting a regulatory feed-forward loop. Pharmacological inhibition of PARP and knock-down of PARP14 completely abrogated the mutant STAT6 gain-of-function phenotype.In summary, our results suggest that PARP14 is a novel target in STAT6 mutant FL. Our data also imply that the biological and clinical impact of STAT6 mutations will heavily depend on the (targetable) upstream activation of the IL-4 signaling cascade, including the abundance of IL-4 / TFH cells in the microenvironment of FL. DisclosuresRichter:HTG Molecular Diagnostics, Inc.: Research Funding. Klapper:Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; HTG Molecular Diagnostics, Inc.: Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Hiddemann:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.

The t(14;18) translocation is absent from endothelial and follicular dendritic cells of follicular lymphoma (FL) and shows heterogeneous presence in preserved FL mantle zones

BackgroundThe translocation t(14;18)(q32;q21) is the genetic hallmark of follicular lymphoma (FL) and can be observed in 85–90% of cases. Whether the translocation is restricted to cells with germinal center B-cell phenotype or can be observed in other cell types of the microenvironment remains debated. Of interest, cases of associated histiocytic and dendritic cell sarcomas arising in the background of FL have been shown to be clonally related and carry the t(14;18), suggesting a “transdifferentiation” of the malignant FL clone into a neoplasm of a different hematopoietic lineage.MethodsWe analyzed the presence of the t(14;18)(q32;q21) as a surrogate marker of the malignant clone in cells of the FL microenvironment using combined fluorescence immunophenotyping and interphase cytogenetics targeting the BCL2 gene locus. In addition to non-lymphoid cells in FL, we analysed FL with preserved IgD+ mantle zones and cases of in situ follicular neoplasia (ISFN) to investigate whether cells of non-germinal center B-cell phenotype are part of the malignant clone.ResultsSix (40%) of 15 manifest FL cases with preserved IgD+ mantle zones did not harbour the t(14;18)(q32;q21) translocation. In all t(14;18) + FL cases, follicular dendritic cells and endothelial cells lacked the t(14;18) translocation. 2/9 FL revealed t(14;18)- IgD+ mantle zone B-cells. In the seven ISFN cases, the t(14;18) translocation was strictly confined to germinal center cells.ConclusionsThe t(14;18) translocation in follicular lymphoma is limited to B-cells. The origin of IgD+ mantle cells is heterogeneous, in the majority of cases belonging to the neoplastic clone, whereas a minority of cases of manifest FL show nonneoplastic mantle zones, similar to ISFN.

Mass cytometry identifies loss of co-stimulatory receptor expression as a novel immune signature in follicular lymphoma

White blood cells in the tumor microenvironment of Follicular Lymphoma (FL) are heterogeneous in phenotype and various subsets have contrasting effect on persistent result. Utilizing Mass Cytometry (CyTOF), we distinguished at any rate 12 subsets of CD4+ T cells in FL biopsy examples and found that a few subsets were increasingly common in FL and less pervasive in tonsil tissue. In particular, we found that CD4+ T cells in FL all the more regularly had a memory phenotype, yet that the quantity of credulous T cells, as opposed to memory cells, was related with a good clinical result. To figure out which memory T cell populaces may adversely influence visualization, we concentrated on 6 subsets of memory cells, two populaces that express CD25 and four that express PD-1. In FL, one of the subsets of CD4+ CD25+ T cells had diminished articulation of CD27 and CD28 and this subpopulation was extended when contrasted with controls. So also, in the PD-1 communicating T-cell subsets, two subsets had diminished articulation of CD27 and CD28 when contrasted with controls. While the complete number of PD-1communicating T cells was not related with FL quiet result, we found that expanded quantities of PD-1+ T cells showing diminished CD27 and CD28 articulation was related with less fortunate patient endurance. We found that T cells with diminished CD27 and CD28 articulation would in general lose articulation of other utilitarian T cell markers, neglected to multiply when invigorated and showed up terminally separated. Moreover, we found that CD70+ lymphoma cells assume a significant job in down-directing articulation of CD27 and CD28 on T cells. Taken together, our mass cytometry results have recognized novel CD4+ memory T cell populaces that are ineffectively practical and are related with a second rate endurance in FL.

The Tumor Microenvironment Is Independently Prognostic of Conventional and Clinicogenetic Risk Models in Follicular Lymphoma

Follicular Lymphoma (FL) is the most common indolent Non-Hodgkin Lymphoma. Despite generally favorable survival outcomes, 20% of FL patients experience ‘Progression of Disease within 24 months’ (POD24) and subsequently have poor long-term overall survival (OS) (Casulo, JCO 2015). Unfortunately, POD24 has limited clinical value because it cannot guide up-front clinical decisions. Accurate pre-therapy prognosticators are vital for clinical trial design and are also increasingly being mandated by funding agencies for stratification of patients to emerging front-line treatments. The new ‘state-of-the-art’ prognosticators ‘m7-FLIPI’ and POD24-PI' (Pastore, Lancet Oncol 2015; Jurinovic, Blood 2016) supplement clinical parameters with genetic mutational status. However, their applicability to population based cohorts including early-stage and asymptomatic patients remains unknown. Furthermore, there is significant heterogeneity of outcome within these prognostic groupings. The established biological and prognostic importance of the tumor microenvironment (TME) in FL suggests that prognosis would be enhanced by incorporating information on host immunity (Scott, Nat Rev Can 2014).Forty-five pre-treatment FL biopsies were categorized into ‘hot’ or ‘cold’ immune nodes by multiplex immunofluorescent imaging and respectively characterized by concordant high or low expression of multiple immune effector and checkpoint-associated proteins. (Fig 1A). Consistent with these findings, gene expression using the Nanostring platform showed that immune effectors (CD4/CD8/TNFa/CD137/CD56) positively correlated with immune checkpoints (PD-1/PD-L1/PD-L2/TIM3/LAG3/CD163/CD68) indicative of an adaptive immune response. Additionally, high-throughput unbiased TCRb sequencing showed the intratumoral TCR repertoire was more clonal in ‘hot’ compared to ‘cold’ FL samples (p=0.024), indicative of a skewed T-cell immune response (Fig 1B).We then applied these findings to an independent population-based cohort of 175 cases of FL from the rituximab era with long-term follow-up (median ~7 years), including advanced (n=137) and localized cases (n=38). The aims were to: a) identify new targetable immune parameters of prognostic importance in the rituximab-era; and b) compare and contrast these with published prognostic tools: FLIPI, FLIPI-2, m7-FLIPI, POD24-PI and ‘immune survival score’ (‘ISS’, Dave, NEJM 2004). OS was not only inferior in those experiencing POD24 (HR 4.88, p<0.0001, Fig 1C) but these patients had a >2-fold increase in 5-year patient health costs. Hence, POD24, as well as FFS and TT2T were therefore chosen as the primary outcome measures.M7 mutation frequencies were similar to those previously published (Pastore, Lancet Oncol 2015). However, the prognostic utility of the m7-FLIPI could not be demonstrated, whereas the FLIPI, FLIPI-2, and POD24-PI retained their prognostic value. The POD24-PI was most predictive of FFS (p<0.0001, HR=3.54) and was most specific in identifying cases that experience POD24 (Sp=68%). The prognostic utility of the TME was then tested. Notably both the ISS (p=0.024, HR=1.74) and multiple immune genes not represented in the ISS including PD-L2, TIM3, LAG3, CD137, TNF and CD4 predicted FFS. PD-L2 demonstrated the strongest association with FFS (p<0.0001, HR=3.74, Fig 1D). It not only out-performed the ISS but was independent to the FLIPI and POD-24-PI. The prognostic significance of PD-L2 was validated in an independent population based cohort of uniformly R-CVP treated patients from an in-silico dataset with gene expression quantified using the Illumina DASL platform (Pastore, Lancet Oncol 2015).We have validated the TME in predicting outcome in a population based cohort of FL patients with long-term follow-up treated in the rituximab era. Furthermore, we describe the role of PD-L2 as well as several additional pertinent, clinically-actionable markers of the TME which predict survival to conventional therapies in FL. Low expression of PD-L2 appears to be a surrogate of a broadly co-ordinated downregulation of the intratumoural response. These immune scores are independent of and additive to additive to the FLIPI and POD24-PI. Development of new prognostic models require the incorporation of host immunity along with clinico-genetic features to further improve the specificity, and to accurately risk stratify FL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Personalized Immunotherapy in Follicular Lymphoma By Intranodal IFN-Dendritic-Cell Combined to Anti-CD20 Antibody

INTRODUCTIONFollicular lymphoma (FL) is considered a chronic, immune responsive and still incurable tumour. Recently two papers have shown the efficacy of active immunotherapy by repeated administration of autologous dendritic cells (DC) in Follicular Lymphoma (FL). Notably, in both studies a third of patients achieved long lasting remission. A novel DC population (IFN-DC), differentiated from human monocytes in the presence of GM-CSF and IFN-alpha has been developed in the laboratory of the Istituto Superiore di Sanità (ISS). IFN-DC have been demonstrated highly efficient in internalizing tumour-cell antigens, recovering "in vitro" T-cell responses from FL patients, as well as in mediating NK cell activation and enhanced cytotoxic effector function toward autologous FL cells. We have recently started a phase I study (approved by EC in 2014, EudraCT: 2013-003158-25) of intranodal immunotherapy based on sequential injection of low-dose anti-CD20 antibody (Ab) and IFN-D, in patients with advanced FL. Here we report early results of the ongoing trial.STUDY DESIGNIFN-DC Trial (EudraCT: 2013-003158-25) is a phase I trial aiming at evaluating safety and tolerability as well as immune and clinical responses of a IFN-DC based therapy in combination with rituximab for the treatment of patients with advanced FL.· The Primary endpoints are: I) Evaluation of safety and tolerability of treatment II) Evaluation of tumor-specific immune responses. by determining: i) Tumor-specific immune response in peripheral blood; ii) Intratumoral infiltration of immune cells; iii) DTH test.· The secondary endpoint is clinical response. The study population consists of patients with relapsed/refractory FL, aged 18-75 years, not needing immediate retreatment basing on the GELF Criteria. The· Treatment: the regimen foresees eight injection cycles, each consisting of the combined administration of rituximab and IFN-DC. The first four administered every two-weeks and the remaining four administered monthly. Rituximab (5-10mg) and low or high doses of IFN-DC (20±5 x106 or 40±5x106cells respectively), are administered by intranodal direct injection (IDI).RESULTSFive patients have been enrolled so far. The treatment proved to be safe, feasible and well tolerated. Three patients have completed treatment and two experienced complete remission with disappearance of both proximal and distal lesions, as confirmed by PET scan. Notably in both patients the distal lymph nodes, which had not been directly treated by intranodal injections, reduced to normal size earlier than those treated by IDI (abscopal effect). One of these two is still in durable complete clinical and molecular remission after 17 months while the latter patient, who had progressed after completing eight cycles of treatment, experienced a complete clinical remission after a second course of immunotherapy ( Fig.1). Two out of three patients performed the DHT test and, despite one of the two achieved remission, they both resulted non-responsive. The characterization of IFN-DC and lymphoma nodes in FL patients have shown high expression of PD-1 and PD-L1 in FL microenvironment and on IFN-DC respectively. Immunomonitoring is currently underway. However, quantification of NF-kB (p65) nuclear translocation by multispectral image flow cytometry, as a marker of NK cell activation, showed the persistence of higher percentages of activated NK cells in the two responding patients (Fig.2).CONCLUSIONCurrently no definitive data prove the benefit of IDI treatment, however the trial is ongoing and other patients are under screening. Notably both the responding patients showed the abscopal effect. The possible set-up of an effective and comparatively economic treatment, that exploits patients' immune system against this chronic and immune responsive tumour, is worth of exploring. Furthermore it can be envisaged that the combination of immune checkpoint inhibitor antibodies to DC-based therapies, might improve clinical activity of DC-based strategies. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Newly diagnosed and relapsed follicular lymphoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Newly diagnosed and relapsed follicular lymphoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Analysis of dendritic cell subpopulations in follicular lymphoma with respect to the tumor immune microenvironment

The immune cell composition of the follicular lymphoma (FL) tumor microenvironment is increasingly recognized as an important determinant for clinical outcome. Here, we explored frequency and distribution of dendritic cell (DC) subtypes in relation to regulatory T cells (Treg) by immunohistochemistry in lymph node biopsies from patients with de novo FL. We found that neoplastic follicles contained lower DC and higher Treg frequencies than hyperplastic follicles in control lymph nodes. Treg numbers particularly correlated with the subset of conventional CD11c+ DCs. Additionally, both a high intra- to interfollicular ratio of CD11c+ DCs and increased intrafollicular Treg frequencies were associated with decreased overall survival. This suggests that functional interactions between these cells may be relevant for FL progression/recurrence. The presence of CD11c+ DCs in the tumor microenvironment may assist tumor infiltration by Tregs, thus contributing to the suppression of an otherwise beneficial T-cell-dominated FL microenvironment.

Follicular Lymphoma: The Role of the Tumor Microenvironment in Prognosis.

The microenvironment of follicular lymphoma (FL) is composed of tumor-infiltrating CD8(+) T cells, follicular regulatory T cells, lymphoma-associated macrophages and mast cells, follicular helper T cells, follicular dendritic cells, and follicular reticular cells, all of which have been reported to have relevance in the prognosis of FL patients. In addition, some of these cells play a role in the histologic transformation of FL. Macrophages contribute to a poor prognosis in FL patients treated in the pre-rituximab era, but are associated with good prognosis in those treated in the rituximab era. T-cell immunoglobulin and mucin domain protein (TIM) 3 are markers of T-cell exhaustion, and T cells co-expressing programed death 1 (PD1) in peripheral blood and lymph nodes secrete interleukin (IL)-12 in the serum. Serum CXCL9, IL-2 receptor, and IL-1 receptor agonist are associated with shorter survival of FL patients. Agents for manipulation of the microenvironment surrounding FL cells include the immunomodulatory drug lenalidomide, immune check-point inhibitors, and cyclophosphamide prior to rituximab. To battle FL and to improve the outcomes of FL patients, understanding the relationship between neoplastic cells and the various microenvironmental cellular components is crucial for developing therapeutics against the microenvironment.

Abstract 3356: Diversity of CD8+ and rgulatory T cells is inversely correlated in follicular lymphoma: A potential predictive biomarker

Abstract Gene expression profiling experiments have demonstrated that the immune microenvironment in follicular lymphoma (FL) plays an important role in controlling the behavior of the disease. FL patients with tumors enriched for T cell-related transcripts have a more favorable prognosis. As T cells have been shown to mediate anti-tumor immunity in other cancers, they may also be critical in controlling the progression of FL. T cells are often identified in and around malignant follicles in FL by immunohistochemistry, but it is not known whether their presence relates to their specificity for antigens expressed by FL cells. Hypothesizing that at least a fraction of T cells in FL are truly lymphoma antigen-specific, we have begun to characterize the T cell receptor (TCR) repertoire of FL tissues using a next generation sequencing platform, and have deeply sequenced T cell receptor (TCR) cDNAs of T cell subset populations present in pretreatment FL biopsy specimens. Regulatory T cell (Treg) TCRs in FL tissues revealed a highly oligoclonal expansion, compared to those in control lymph nodes. Furthermore, an inverse correlation was observed between the diversity of Treg and CD8+ TCRs in FL specimens. Interestingly, a tumor from an FL patient, who has received no anticancer treatment for more than 10 years, was found to have missense mutations in the peptide-binding domain of both HLA class I and class II molecules, which might have presented tumor-specific antigens and enhanced host immune responses. Although further verification is required, our data suggest that the T cell repertoire is skewed and restricted in FL and support the evolving understanding of the microenvironment in this disease. Citation Format: Justin Paul Kline, Xiao Liu, Girish Venkataraman, Jiaying Lin, Kazuma Kiyotani, Sonali M. Smith, Magdeline Montoya, Yusuke Nakamura. Diversity of CD8+ and rgulatory T cells is inversely correlated in follicular lymphoma: A potential predictive biomarker. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3356. doi:10.1158/1538-7445.AM2015-3356

XIV. The pathology of transformation of indolent B cell lymphomas.

XIV. The pathology of transformation of indolent B cell lymphomas.

Highly clonal regulatory T-cell population in follicular lymphoma – inverse correlation with the diversity of CD8+ T cells

The immune microenvironment in follicular lymphoma (FL) plays an important role in controlling disease characteristics. To characterize the T-cell receptor (TCR) repertoire in follicular lymphoma (FL) tissues, we applied a next-generation sequencing platform and deeply sequenced TCR cDNAs of T-cell subset populations present in pretreatment FL biopsy specimens. T regulatory cell (Treg) TCRs in FL tissues revealed a highly oligoclonal expansion compared to those in control lymph nodes. Furthermore, an inverse correlation was observed between the diversity of Treg and CD8+ TCRs in FL specimens. Interestingly, a tumor from an FL patient, who had not received anticancer treatment for more than 10 years, was found to have missense mutations in the peptide-binding domain of both human leukocyte antigen (HLA) class I and II molecules, which might have presented tumor-specific antigens and enhanced host immune responses. Although further verification is required, our data suggest that the T-cell repertoire is skewed and restricted in FL and support the evolving understanding of the microenvironment in this disease.

Abstract A24: Investigation of the spatial heterogeneity of specific immune cell phenotypes in the tumor microenvironment of follicular lymphoma

Abstract Background. Tumor-infiltrating lymphocytes (TILs) are present in the tumor microenvironment of many cancers, with consequent tumor immunogenicity and association with survival. In particular, increased levels of regulatory T cells (Tregs) are associated with poorer prognosis in some cancers. However, given the complexity of TIL phenotype their visualization in situ is difficult, and impossible without multiplex immunophenotyping. An understanding of both the phenotype and spatial distribution of TILs in situ within the tumor microenvironment would be advantageous to understanding their role in tumour immunobiology, especially given growing interest in tumour immunotherapy. Here we present a multi-marker, computer-aided method for analysing the distributions of CD3/FOXP3 (Treg) and CD3/CD69 (Tact) T cells in follicular lymphoma sections using a multispectral imaging (MSI) and automated analysis approach. An hypothesized interaction distance (HID) analysis was used to determine whether the spatial patterns of Tregs and Tacts were prognostically significant Design. A single section of a tissue microarray containing triplex follicular lymphoma cores from 40 subjects [24 male, 16 female, age 35 to 75 years at diagnosis, median 55 years , 2- 171 months follow-up] was stained for CD3, FOXP3, CD69 and hematoxylin. Each core was imaged using MSI and the individual staining of each marker separated from each other using spectral unmixing. CD3+ TILs were located using automated image analysis. The FOXP3 and CD69 status of each CD3+ TIL was then determined and the spatial distributions of the CD3/FOXP3 and CD3/CD69 cells were used as input into the HID analysis. Results. Multiplexed IHC staining, MSI and automated per-cell quantitative analysis was successful. Kaplan-Meier analysis demonstrated favourable outcome with higher numbers of CD3+, CD3+/FOXP3+ and CD3+/CD69+ cells. HID analysis demonstrated the association of favourable outcome with a high entropy , representative of a diffuse spatial pattern, of FOXP3+ and CD69+ positive T cells. Conclusion. In this study we report that higher Treg cell counts in a diffuse pattern was associated with favorable prognosis. This supports the importance of Tregs in the tumour microenvironment. It is pertinent to mention that contradictory findings are routinely reported from studies investigating the role of Tregs in solid and haematological malignancies. This is due to the complex interactions between pro-/anti- tumour immune factors present in the tumour microenvironment. The resultant effects are due to the summation of the activities of these factors. It is therefore even more relevant that a method such as exhibited here, capable of defining and measuring the effect on patient outcome of the spatial patterns of multiple cellular phenotypes in the tumor microenvironment is available. Citation Format: Lilli Nelson, James Mansfield, Roslyn Lloyd, Chris van der Loos, Chris Rose, Richard Byers. Investigation of the spatial heterogeneity of specific immune cell phenotypes in the tumor microenvironment of follicular lymphoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A24. doi:10.1158/1538-7445.CHTME14-A24

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

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

Intratumoral and Peripheral Blood T Cells Recognize Mutated Neo-Antigens in Follicular Lymphoma

Introduction: The tumor microenvironment of follicular lymphoma is heavily infiltrated with CD4+ and CD8+ T cells that are tumor-reactive and produce cytokines such as IFN-g, TNF-a, and GM-CSF that may play a role in tumor control or regression. However, the nature of the tumor antigen(s) recognized by these T cells is unknown. Here, we investigated the possibility that intratumoral T cells recognize mutated tumor antigens, which may be recognized as neo-antigens by the host immune system as opposed to non-mutated proteins that are recognized as self-antigens.Methods: Whole exome sequencing was performed on three follicular lymphoma tumors and their corresponding germline (peripheral blood mononuclear cells, PBMC) samples obtained at initial diagnosis. Nonsynonymous mutations were identified. The binding affinities of the peptides spanning each mutation to their respective MHC class I and class II molecules were determined in silico by MHC binding algorithms. Peptides predicted to have high binding affinity (IC50<500 nM) were selected for synthesis. For MHC Class I binding peptides, nonamer peptides predicted to bind to HLA-A*0201 (all three follicular lymphoma patients expressed HLA-A*0201) were synthesized and for MHC Class II binding peptides, 40-mer polypeptides were synthesized with the mutated amino acid in the middle. Corresponding wild type peptides were synthesized where necessary. Autologous T cells isolated from PBMC or tumors obtained at initial diagnosis were used to test reactivity to mutated and/or wild type peptides. Autologous PBMCs depleted of T cells were used as antigen-presenting cells in co-culture assays with peripheral blood or intratumoral T cells. IFN-g was assessed in culture supernatants by ELISA.Results: In patient 1, of 61 nonsynonymous mutations, 9 nonamer peptides and 16 pentadecamer peptides were predicted to bind with high affinity to HLA-A*0201 and HLA Class II alleles expressed in the patient, respectively. Of the 9 nonamer HLA-A*0201-binding peptides, two were recognized by autologous peripheral blood and intratumoral T cells. Of the 16 HLA Class II-binding peptides, autologous peripheral blood and intratumoral T cells recognized four. HLA Class II and HLA-DR blocking antibodies but not HLA Class I and HLA-DP or DQ blocking antibodies inhibited production of IFN-g in response to the HLA Class II-binding peptides suggesting that they were recognized by CD4+ T cells. More importantly, the T cells were not reactive against the wild type peptides tested suggesting that the mutations converted the non-immunogenic wild type proteins into immunogenic neo-antigens. Testing of mutated peptides from additional patients is ongoing.Conclusions: Our results suggest that peripheral blood and intratumoral T cells recognize mutated neo-antigens in follicular lymphoma. These results are consistent with recent results from solid tumors that showed intratumoral CD4+ and CD8+ T cells primarily recognize mutated neo-antigens. More importantly, administration of such neo-antigen-reactive CD4+ and CD8+ T cells as adoptive T-cell therapy resulted in long-term tumor regressions in humans. Thus, identification of immunogenic neo-antigens may facilitate development of novel personalized forms of cancer immunotherapy including therapeutic vaccines and adoptive T-cell therapy strategies. Moreover, characterization of such cancer regression antigens will be valuable to understand the effects of immune checkpoint antagonists and other novel immunotherapies. DisclosuresNo relevant conflicts of interest to declare.