Malignant Lymphocytes Research Articles

FilGAP, a Rac-specific Rho GTPase-activating protein, is a novel prognostic factor for follicular lymphoma.

FilGAP, a Rho GTPase-activating protein (GAP), acts as a mediator of Rho/ROCK (Rho-associated protein kinase)-dependent amoeboid movement, and its knockdown results in Rac-driven mesenchymal morphology. Herein, we focus on the possible roles of FilGAP expression in normal and malignant lymphocytes. Eighty-three cases of follicular lymphoma (FL), 84 of diffuse large B-cell lymphoma (DLBCL), and 25 of peripheral T-cell lymphoma (PTCL), as well as 10 of normal lymph nodes, were immunohistochemically investigated. In normal lymph nodes, FilGAP immunoreactivity was significantly higher in lymphocytes in the mantle zone as compared to those in the germinal center and paracortical areas. In contrast, the expression levels of both cytoplasmic and perinuclear Rac1 were significantly lower in the germinal center as compared to paracortical regions, suggesting that changes in the FilGAP/Rac axis may occur in B-cell lineages. In malignant lymphomas, FilGAP expression was significantly higher in B-cell lymphomas than PTCL, and the immunohistochemical scores were positively correlated with cytoplasmic Rac1 scores in FL and DLBCL, but not in PTCL. Patients with FL and germinal center B-cell-like (GCB)-type DLBCL showing high FilGAP scores had poor overall survival rates as compared to the low-score patients. Moreover, multivariate Cox regression analysis showed that a high FilGAP score was a significant and independent unfavorable prognostic factor in FL, but not in DLBCL. In conclusion, FilGAP may contribute to change in cell motility of B-lymphocytes. In addition, its expression appears to be useful for predicting the behavior of B-cell lymphoma, in particular FL.

Opposing regulation of BIM and BCL2 controls glucocorticoid-induced apoptosis of pediatric acute lymphoblastic leukemia cells

AbstractGlucocorticoids are critical components of combination chemotherapy regimens in pediatric acute lymphoblastic leukemia (ALL). The proapoptotic BIM protein is an important mediator of glucocorticoid-induced apoptosis in normal and malignant lymphocytes, whereas the antiapoptotic BCL2 confers resistance. The signaling pathways regulating BIM and BCL2 expression in glucocorticoid-treated lymphoid cells remain unclear. In this study, pediatric ALL patient-derived xenografts (PDXs) inherently sensitive or resistant to glucocorticoids were exposed to dexamethasone in vivo. Microarray analysis showed that KLF13 and MYB gene expression changes were significantly greater in dexamethasone-sensitive than -resistant PDXs. Chromatin immunoprecipitation (ChIP) analysis detected glucocorticoid receptor (GR) binding at the KLF13 promoter to trigger KLF13 expression only in sensitive PDXs. Next, KLF13 bound to the MYB promoter, deactivating MYB expression only in sensitive PDXs. Sustained MYB expression in resistant PDXs resulted in maintenance of BCL2 expression and inhibition of apoptosis. ChIP sequencing analysis revealed a novel GR binding site in a BIM intronic region (IGR) that was engaged only in dexamethasone-sensitive PDXs. The absence of GR binding at the BIM IGR was associated with BIM silencing and dexamethasone resistance. This study has identified novel mechanisms of opposing BCL2 and BIM gene regulation that control glucocorticoid-induced apoptosis in pediatric ALL cells in vivo.

Collective Cell Motility Promotes Chemotactic Prowess and Resistance to Chemorepulsion

Collective cell migration is a widespread biological phenomenon, whereby groups of highly coordinated, adherent cells move in a polarized fashion. This migration mode is a hallmark of tissue morphogenesis during development and repair and of solid tumor dissemination. In addition to circulating as solitary cells, lymphoid malignancies can assemble into tissues as multicellular aggregates. Whether malignant lymphocytes are capable of coordinating their motility in the context of chemokine gradients is, however, unknown. Here, we show that, upon exposure to CCL19 or CXCL12 gradients, malignant B and T lymphocytes assemble into clusters that migrate directionally and display a wider chemotactic sensitivity than individual cells. Physical modeling recapitulates cluster motility statistics and shows that intracluster cell cohesion results in noise reduction and enhanced directionality. Quantitative image analysis reveals that cluster migration runs are periodically interrupted by transitory rotation and random phases that favor leader cell turnover. Additionally, internalization of CCR7 in leader cells is accompanied by protrusion retraction, loss of polarity, and the ensuing replacement by new leader cells. These mechanisms ensure sustained forward migration and resistance to chemorepulsion, a behavior of individual cells exposed to steep CCL19 gradients that depends on CCR7 endocytosis. Thus, coordinated cluster dynamics confer distinct chemotactic properties, highlighting unexpected features of lymphoid cell migration.

Common variable immunodeficiency (CVI): case report

The CVI is the second most common primary immunodeficiency. The prevalence of CIV in the world is approximately 1:25,000. The age of presentation of ICV has peaks in the first decade if life and the beginning of the third decade. Both sexes are affected. Diagnostic criteria according to ESID/PAGID are: - recurrent infections; -age more 4 years; -reduced levels of IgG; decrease of IgA and/or IgM; exclusion of the other causes of hypogammaglobulinemia; - no isohemaglutininas and response to the vaccine. Five clinical phenotypes are described: only infections, autoimmunity, polyclonal lymphocytic infiltration, malignancy and enteropathy. The treatament of patients with CVI is performed with the use of an infusion of immunoglobulin.

In Vitro and in Vivo oncolytic Activity of Lasota Strain of Newcastle Disease Virus on a Lymphoma B-Cell Line and a Canine Cutaneous T-Cell Lymphoma

Oncolytic viruses, either naturally or genetically modified, possess the ability to kill cancer cells. Newcastle Disease Virus (NDV), a type I avian paramyxovirus, has demonstrated a selective ability to kill cancer cells directly as well as through immunostimulation. NDV is able to infect more than 250 species of birds, particularly poultry and is considered a zoonosis, primarily causing conjunctivitis. Importantly, no infections have been reported in mammalian species nor is there evidence of human to human transmission. We use the LaSota strain which is lentogenic (less pathogenic). We seek to study the in vitro oncolytic (lympholytic) potential of NDV on healthy mononuclear and malignant lymphocytes and the in vivo oncolytic therapeutic potential in dogs with lymphoma.LaSota vaccine strain (LSV) was propagated in pathogen –free 9 day old chicken embrios. Viral titers were reported as mean infective dose. Healthy human and canine mononuclear cells as well as a human diffuse large B cell lymphoma cell line (DHL4 cell line) were exposed to medium alone, LSV at a multiplicity of infection (MOI) of 0.1, 10 and 100 or CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy. Apoptosis was assessed by flow cytometry using annexin V/FITC/IT. Cell counts were carried out at 48 and 72 hs. Data was analyzed by FlowJo 9 and Prisma 6 Softwares. Interestingly, cells at the highest MOI (100) had the same rate of apoptosis as those exposed to chemotherapy, both significantly higher than negative controls.The in vivo response and toxicity to intravenous and intratumoral inyection of LSV in a dog with naturally occurring aggressive, multicentric cutaneous T-cell lymphoma is reported. The patient had a 4 week history of paraplegia due to lymphomatous infiltration of the spinal canal and a 3 week history of rapid development of multiple elevated cutaneous lesions ranging from (from 0.5 by 0.5 cm to 15 cm in diameter and 2 cm in thickness). After informed consent, LSV was given in a single occasion with 1x106 mean (50%) infective dose in tissue culture (TCDI 50) in only one lesion, as well as intravenously with a TCDI 50 of 1x1012 in order to assess response of distant non-virally infiltrated lesions. At four weeks of follow up, no new lesions have appeared. All tumors are responding with complete flattening of the lesions and decrease in diamenter of all lesions. Spontaneous bleeding from the lesions was seen from days 4 through 7 after treatment. All laboratory work up is within normal limits except for indirect hiperbilirrubinemia (2x above normal) in the 4th week (Coombs results are pending). Consecutive samples for histology, electron microscopy and viral load are being evaluated. The patient has had an excellent performance status with no evident clinical toxicity and has begun to wag his tail and show slow movement of the posterior limbs with gain in sensitivity.We have demonstrated the specific in vitro, oncolytic/lympholytic activity of NCV (LaSota) on human lymphoma- derived cells with minimal tropism towards healthy cells. Response to treatment of the first patient (dog) is encouraging. Longer follow up as well as histologic evaluation and viral shedding are being evaluated. DisclosuresNo relevant conflicts of interest to declare.

Investigating a New Methodology for the Detection of Low Frequency 17p Deleted Clones in Chronic Lymphocytic Leukaemia (CLL): Impact on Prognostic Outcome and Therapeutic Resistance

Cytogenetic analysis by standard FISH of malignant CLL lymphocytes has been identified as predictive of disease outcome. However CLL is likely to be a disease of many sub-clones which either evolve post chemo-immunotherapy or are present in very low frequency at the outset. In the contemporary therapeutic environment greater understanding of these drug resistant sub-clones is required to prevent inappropriate treatment options being employed to treatment naive patients. However, this can only be achieved by meaningful and reliable analysis of low frequency clones.CLL patients with 17p (TP53 gene) deletion, have the poorest outcome with an overall survival of 2-3 years due to chemo-refractoriness and early relapse. Although TP53 gene deletions are infrequent in CLL at initial diagnosis (5%-10%), their frequency increases to 40%-50% in the relapsed refractory patients. Importantly, CLL patients having less than 5% 17p deleted cells by standard FISH are considered normal with good prognosis, while patients with high frequency 17p deleted nuclei have the worst outcomes. A significant number of patients have TP53 deleted clones of the order of 5-40% of total cell number in which we suspect chemotherapy will have a disadvantageous effect on the genetic profile but whom currently receive this treatment.AIMS/METHODS: We are therefore exploring techniques to reliably quantify low frequency clones carrying 17p and other critical gene deletions using Flow FISH a flow cytometry-based method (Amnis Corp) capturing multiple images by Image Stream X (ISX). Extended Depth of Field (EDF) technology expands the depth of focus over the entire cell to enumerate nuclear FISH probe “spots” accurately. The "spot count/cell” program allows to stratify the number of “spots” which based on the intensity and size parameters counted for 10000 cells. Our current project requires development of unique probe sets with sufficient signal to allow reliable detection of single gene deletions with this technology.Data obtained with centromeric probes allows separation of single chromosome deletion and separation of 1 spot vs. 2 spots on image analysis. RP11 BAC probes have been selected for the region of 17p loss and individually nick translated. Individual probes have been tested by standard FISH analysis for repetitive DNA and region specificity and then applied in multiplex to intensify signal. Orange channel fluoresce has been selected to eliminate background fluorescence from histones. Preliminary results have demonstrated satisfactory technical outcomes of signal over background for utilization of this approach. [Display omitted] [Display omitted] [Display omitted] DisclosuresKuss:Roche: Research Funding; Sanofi: Research Funding.

Obinutuzumab (GA101) Is Less Prone to Antagonism of Immune Effector Function By Ibrutinib Than Rituximab in Vitro and in Vivo

Abstract Introduction: Kohrt et al., Blood, 2014 demonstrated that ibrutinib antagonizes ADCC function of rituximab in vitro in ADCC assays and in vivo in the DHL-4 xenograft model through inhibition of FcgammaR signaling in immune effector cells, possibly mediated by inhibition of ITK. Obinutuzumab (GA101) is a glycoengineered type II CD20 antibody that mediates higher direct cell death induction than rituximab, and by being glycoengineered mediates enhanced induction of ADCC and ADCP. Here we aimed to investigate the impact of ibrutinib on the immune effector function of obinutuzumab as compared to rituximab. Experimental methods: The impact of ibrutinib (dose range 30, 100, 300 ng/ml to cover Cmax and Ctrough in patients) on NK cell mediated ADCC induction by obinutuzumab and rituximab was investigated using SU-DHL4 and Z138 cells as targets in LDH and chromium release assays or measuring CD16 downmodulation and the degranulation marker CD107a. IFNg release as a surrogate for NK cell activation was investigated using DHL-4 target cells or an autologous in vitro system using leukemic cells derived from CLL/NHL patients. Depletion of CD19 positive B-cells was determined in whole blood from healthy volunteers in flow cytometry-based whole blood assay. In vivo the combination of obinutuzumab or rituximab (10 mg/kg once weekly for 3 weeks) with ibrutinib (25mg/kg BID days 14-28) was investigated in the DHL-4 xenograft model. Results: In ADCC assays, ibrutinib (dose range 30, 100, 300 ng/ml) resulted in a reduction of the ADCC potency of obinutuzumab and rituximab. However, at saturating antibody concentrations of 10 ug/ml, ADCC mediated by obinutuzumab was retained while ADCC mediated by rituximab was strongly reduced as measured by chromium release (Figure 1A). Interestingly, in the whole blood B cell depletion assay only little impact of ibrutinib on obinutuzumab-mediated B cell depletion in terms of EC50 and maximal killing was observed at clinically meaningful concentrations of ibrutinib (30, 100, 300 ng/ml), while the activity of rituximab could be completely abolished with 300 ng/ml ibrutinib (Figure 1B). Notably, control experiments using an effector dead version of obinutuzmab that cannot any longer mediate ADCC or ADCP demonstrate that the retained B cell depletion by obinutuzumab in presence of ibrutinib is not due to direct cell death induction, but also due to immune effector cell mediated function (ADCC and ADCP). In the DHL-4 xenograft model where ibrutinib as a single agent has no anti-tumoral efficacy, the combination resulted in a reduced anti-tumoral efficacy of rituximab, whereas efficacy of obinutuzumab was not affected (Figure 1C). Conclusions: Surprisingly, we found that the inhibitory effect of ibrutinib on the immune effector mediated activity of obinutuzumab is not observed when compared to rituximab. Most notably, ADCC at saturating antibody doses, whole blood B cell depletion and in vivo efficacy of obinutuzumab were retained in presence of clinically relevant concentrations of ibrutinib covering Cmax and Ctrough levels, whereas the activity of rituximab was almost completely abolished under these conditions. We hypothesize that the differential behavior of obinutuzumab and rituximab may be related to the enhanced FcgRIII affinity and stronger FcgRIII signaling activation mediated by obinutuzumab as a consequence of glycoengineering that may subsequently overwrite inhibitory effects of ibrutinib. While the clinical relevance of the observed preclinical antagonism for the combination of rituximab with ibrutinib still needs further clinical investigation, these preclinical data strongly support the clinical investigation of ibrutinib in combination with the glycoengineered Type II CD20 antibody obinutuzumab for the treatment of chronic lymphocytic leukemia and other B-cell malignancies. Figure 1 Figure 1. Disclosures Herter: Roche: Employment. Bacac:Roche: Employment. Umana:Roche: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

CXCL12 Enhances CLL Cell and T-Cell Migration in a Dynamic Circulating Model of CLL That Can be Abrogated By the CXCR4 Antagonist ONO-7161

We have recently developed a circulating model of CLL that more accurately mimics the transient interactions that take place between lymphocytes and the vascular endothelium. Normal and malignant lymphocytes actively undergo transendothelial migration in our system in the absence of a chemoattractant. We have now refined our model to include a CXCL12 gradient and evaluated the ability of CXCR4 antagonists to modulate lymphocyte migration. Initially we demonstrated that a single deposition of CXCL12 (100ng/ml) into the extravascular space (EVS) of our model significantly increased CLL cell migration (P = 0.049, mean circulating CXCL12 level = 254pg/ml). Subsequently, we created a stable chemokine gradient by seeding CXCL12-secreting MRC5 cells into the EVS. Although this resulted in lower peak circulating levels of CXCL12 (as measured by ELISA, mean 23pg/ml), it significantly increased migration (P = 0.024) presumably because of the constant nature of the chemokine gradient. As previously shown CXCR4 expression is up-regulated on circulating CLL cells when compared to static cultures (P=0.023). Basal CXCR4 expression and expression after circulation for 48h, correlated with the degree of CLL cell migration into the EVS (r2=0.40, P=0.003; r2=0.31, P=0.009 respectively). It is worthy of note that as a proportion of all circulating peripheral blood mononuclear cells, CD3+ T-cells migrated significantly more than CD19+ CLL cells (T-cells: 12.72% ±13.47, CLL cells: 1.16% ±1.23 P =0.02, n = 10). This may be partly the result of the known defects in CLL cell migration but is also likely to be promoted by CLL cell secretion of T-cell attracting chemokines CCL3 (298pg/ml ±197.5, n=13) and CCL4 (56pg/ml ±115.6, n=13) in our model. Furthermore, migrated T-cells and CLL cells recovered from the EVS showed a marked increase in Ki-67 expression (P<0.0001 and P<0.0001 respectively) confirming that both subsets of cells are activated. We next explored whether we could block the CXCR4/CXCL12 axis using the CXCR4 antagonist ONO-7161. In transwell chambers (3μm pores) 30nM ONO-7161 resulted in significantly decreased CLL cell migration (54.1% ±25.53 of control, P=0.0003) and was more potent than Plerixafor (1μM) (P=0.06). Additionally, in the circulating system, ONO-7161 resulted in significantly reduced CLL cell migration into the EVS at 48h (P=0.0006). T-cell migration was also reduced by ONO-7161 but not significantly. In summary, we have enhanced our circulating model system of CLL by adding a physiologically relevant CXCL12 chemokine gradient. This then allowed us to dissect the role of the CXCR4/CXCL12 axis in modulating CLL cell migration. Finally we demonstrated that the CXCR4 antagonist ONO-7161 was a potent inhibitor of CLL cell migration in our model. Given that ONO-7161 was non-toxic to CLL cells at concentrations up to 10μM, it seems likely that it might be usefully combined with chemotherapy and biologically targeted agents in the treatment of this and other diseases. DisclosuresYoshizawa:Ono Pharmaceutical Co., Ltd.: Employment. Fegan:ONO PHARMA: Honoraria.

Novel Approach to the Potential Treatment of Patients with B-Cell Lymphomas Harboring the MYD88 L265P Mutation: Combination Treatment with TLR Antagonist and Rituximab

Introduction. Patients with B-cell lymphomas harboring the oncogenic MYD88 L265P mutation have poor response to standard of care regimens such as R-CHOP (Fernandez-Rodriguez et al, Leukemia, 2014, Jun 6). These patients also have not responded well to new therapies under development, including BTK inhibitors (Wilson et al, Blood 2012; 120). The MYD88 L265P oncogenic mutation has been shown to over-activate TLR7- and TLR9-mediated signaling pathways, including NF-κB, IRAK1/4 and STAT3, which in turn promote cell survival and proliferation (Lim et al., AACR 2013, Abstract #2332). Our approach to a potential treatment of B-cell lymphomas harboring the MYD88 L265P mutation is to block TLR7- and TLR9-mediated signaling through use of a TLR antagonist. IMO-8400 is an antagonist of TLR7, TLR8, and TLR9 that inhibits cell signaling and induces apoptosis specifically in B-cell lymphoma cell lines harboring the MYD88 L265P mutation (Bhagat et al, AACR 2014, Abstract #2570). IMO-8400 is currently in clinical development for the treatment of relapsed/refractory patients with Waldenström’s macroglobulinemia (WM) and diffuse large B-cell lymphoma (DLBCL).Methods. The current preclinical studies were designed to evaluate the combination of IMO-8400 with rituximab, which is a key component of R-CHOP. The studies were conducted in xenograft models of the activated B-cell like (ABC) DLBCL cell line OCI-Ly10 and the WM cell line MWCL-1, both positive for the MYD88 L265P mutation. For the ABC-DLBCL model, OCI-Ly10 cells were implanted s.c in NOD-SCID mice on day 0 and treatment was initiated on day 9 when mean tumor volume reached ~200 mm3. For the WM model, MWCL-1 cells were implanted s.c. in NOD-SCID mice on day 0 and treatment was initiated on day 6 when mean tumor volume reached ~250 mm3. In both models, the tumor-bearing mice were treated with PBS (control group), IMO-8400 (25 mg/kg, i.p.), rituximab (10 mg/kg, i.p.) or a combination of the two agents. IMO-8400 was administered twice per week for 3 weeks and continued further as maintenance treatment once per week. Rituximab was administered twice per week for three weeks in the ABC-DLBCL model whereas it was given only on days 3, 6, and 9 in the WM model.Results. Treatment of tumor-bearing animals with IMO-8400 or rituximab alone and in combination was well tolerated in both the models. In the ABC-DLBCL model, tumor growth inhibition compared to PBS control was 91.3% in the combination therapy group vs. 40.8% with IMO-8400 alone (p<0.001) and 51.6% with rituximab alone (p=0.003). Inhibition of tumor growth was associated with decreases in levels of several tumor-secreted circulating human cytokines including IL-10 (p<0.05 vs control) in all the three treatment regimens. In the WM model, tumor growth inhibition was 87% in combination group vs. 47% with IMO-8400 (p<0.001) and 72% with rituximab (p<0.001). Further, tumor growth inhibition in the WM model was associated with reduction in serum IgM levels (p<0.03 vs control) in the single-agent as well as the combination treatment groups. Importantly, combination therapy resulted in complete tumor regression in more than 80% of mice in both ABC-DLBCL and WM models. Histological analysis of the residual tumors showed disappearance of malignant lymphocytes with replacement by fibrotic stroma.Conclusions. Our data show that IMO-8400 in combination with rituximab was well tolerated in mouse xenograft models and exerted potent anti-tumor activity against B-cell lymphomas with the MYD88 L265P oncogenic mutation. These observations provide a strong rationale to evaluate IMO-8400 in combination with first-line rituximab-based therapies for the treatment of patients with B-cell lymphomas harboring the MYD88 L265P mutation. DisclosuresWang:Idera Pharmaceuticals: Employment. Jiang:Idera Pharmaceuticals: Employment. Sullivan:Idera Pharmaceuticals: Employment. Bhagat:Idera Pharmaceuticals: Employment.

Treatment of Stage IA Nodular Lymphocyte-Predominant Hodgkin Lymphoma (NLPHL): An Analysis from the German Hodgkin Study Group (GHSG)

Introduction: Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma entity accounting for about 5% of all HL cases. As compared with classical HL (cHL), NLPHL is characterized by the absence of CD30 and the consistent expression of CD20 on the malignant lymphocyte predominant (LP) cells. Given a more indolent clinical course, especially early-stage NLPHL is often treated less aggressive than classical HL (cHL). In stage IA patients, radiotherapy (RT) alone is applied at most institutions. However, this clinical practice is not based on data from prospective clinical trials with sufficient follow-up. To shed more light on the optimal treatment of stage IA NLPHL, we performed an analysis including patients with long-term follow-up treated within German Hodgkin Study Group (GHSG) clinical trials.Patients: A total of 256 stage IA NLPHL patients treated within 7 prospective GHSG studies between 1988 and 2009 were included in the analysis. Treatment consisted of combined-modality treatment (CMT) (n=72), extended-field RT (EF-RT) (n=49), involved-field RT (IF-RT) (n=108) or four weekly doses of the anti-CD20 antibody rituximab (n=27).Results: The median age at NLPHL diagnosis was 38.5 years (range: 17-75); 194/256 (75.8%) patients were male and 62/256 (24.2%) patients were female. Median follow-up for the whole patient group was 91 months (98 months for CMT, 118 months for EF-RT, 87 months for IF-RT, 49 months for rituximab). All patients responded to treatment irrespective of the treatment modality applied. At 8 years, progression-free survival (PFS) and overall survival (OS) rates were 88.5% and 94.5% for CMT, 84.3% and 95.7% for EF-RT and 91.9% and 99.0% for IF-RT; 4-year PFS and OS rates for patients treated with rituximab were 81.0% and 100%. Seventeen patients developed a second malignancy in the course of follow-up (8 after CMT, 3 after EF-RT, 4 after IF-RT, 2 after rituxmab). Nine of these second malignancies were solid tumors (4 after CMT, 2 after EF-RT, 1 after IF-RT, 2 after rituximab) and 8 were hematologic malignancies (4 after CMT, 1 after EF-RT, 3 after IF-RT, none after rituximab). A total 12 deaths occurred. The most common cause of death was cardiac failure (n=3). Only one patient died from NLPHL.Conclusion: Based on this large analysis with long-term follow-up, IF-RT should be the standard of care for stage IA NLPHL. Treatment with single agent rituximab is associated with an increased event rate when compared with IF-RT and should therefore not be routinely used in stage IA NLPHL patients. However, given the shorter follow-up in comparison with CMT, EF-RT and IF-RT, final conclusions regarding rituximab especially in terms of treatment-related late sequelae cannot yet be drawn. Addition of chemotherapy does not improve the excellent outcome achieved with RT alone. DisclosuresOff Label Use: Rituximab in NLPHL.

Molecular characterization of subcutaneous panniculitis-like T-cell lymphoma reveals upregulation of immunosuppression- and autoimmunity-associated genes

BackgroundSubcutaneous panniculitis-like T cell lymphomas represent a rare and difficult to diagnose entity of cutaneous T cell lymphomas. SPTL affects predominantly young adults and presents with multifocal subcutaneous nodules and frequently associated autoimmune features. The pathogenesis of SPTL is not completely understood.MethodsThe aim of this study was to unravel molecular pathways critical to the SPTL pathogenesis. Therefore, we analyzed 23 skin samples from 20 newly diagnosed SPTL patients and relevant control samples of adipose and non-malignant panniculitis tissue by using gene expression microarray, quantitative PCR, and two-colour immunohistochemistry.ResultsInterestingly, indoleamine 2,3-dioxygenase (IDO-1), an immunotolerance-inducing enzyme, was among the most highly overexpressed genes in all comparisons. The expression of Th1-specific cytokines, known to be associated with autoimmune inflammation (i.e. IFNG, CXCR3, CXCL9, CXCL10, CXCL11, and CCL5), were also significantly increased. Confirmed using immunohistochemistry, the morphologically malignant lymphocytes expressed CXCR3 and CXCL9. IDO-1 expression was found both in some morphologically malignant lymphocytes rimming the adipocytes and in surrounding CD11c− CD68− cells but not in CD11c+ dendritic cells in the microenvironment. The proportion of FoxP3+ cells in SPTL exceeded that in the benign panniculitis samples.ConclusionsOur results indicate that the up regulation of the tolerogenic IDO-1 together with the up regulation of IFNG, CXCR3 ligands, and CCL5 are features of SPTL lesions. We anticipate that the IFNG-inducible IDO-1 expression contributes to the formation of an immunosuppressive microenvironment, favorable for the malignant T cells. This study provides a relevant molecular basis for further studies exploring novel therapeutic means for subcutaneous T cell lymphoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0160-2) contains supplementary material, which is available to authorized users.

Abstract A07: Phase I evaluation of anti-human leukocyte antigen-DR monoclonal antibody therapy in spontaneous canine lymphoma

Abstract IMMU-114 (also known as hL243) is a humanized anti-HLA-DR monoclonal antibody (mAb) with potent anti-tumor activity against a broad range of human hematopoietic malignancies. As MHC Class II antigens are also expressed on canine lymphocytes, IMMU-114 has previously been found to recognize and kill malignant lymphocytes collected from dogs with spontaneous B cell lymphoma. Canine lymphoma is a highly relevant animal model of B-cell non-Hodgkin's lymphoma (NHL) in humans with many advantages over rodent models. In addition, clinical trials in pet dogs can be designed to address therapeutic endpoints relevant to phase II/III studies in both dogs and humans. This study is being undertaken to determine the safety, pharmacodynamic and pharmacokinetic profiles of IMMU-114 in dogs with heavily pre-treated B cell lymphoma (NHL). Our hypothesis is that the mAb can safely be administered to tumor-bearing dogs and that it will be preferentially cytotoxic to B lymphocytes. Our overall objectives are to characterize the toxicity and biologic profile of IMMU-114 to advance the therapy of canine lymphoma and to better inform human clinical trials using anti-HLA-DR mAbs. Thirteen pet dogs with B cell lymphoma demonstrating binding to IMMU-114 have been enrolled in an on-going phase I clinical trial at the Flint Animal Cancer Center at Colorado State University. Enrollment follows a standard 3 x 3 dose escalation to identify the maximally tolerated dose (MTD) of the mAb, starting at a dose of 3 mg/kg, and escalating thus far to 6, 12, and 24 mg/kg. Data collection includes pre and post-infusion peripheral blood mononuclear cells (PBMCs), serum, serial lymph node measurements, complete blood counts and serum chemistry profiles. Preliminary pharmacodynamic evaluation shows a significant and transient dose-dependent decrease in the percentage and absolute number of B lymphocytes in the blood of treated dogs 24 hours post infusion. This corresponds to a transient increase in the relative percentage of T lymphocytes. Neutropenia and other bone marrow toxicities have not been observed. Infusion related side effects appear common but have been mild to moderate in nature with 10/13 patients experiencing grade 1 or grade 2 allergic reactions (fever, urticaria, and hypersalivation) and 2/13 with grade 1 nausea. No dose limiting toxicities have been observed; therefore the MTD has not yet been reached. Thus far 2/13 dogs (15%) have had prolonged disease stabilization and received multiple weekly infusions of 12 mg/kg while 11/13 dogs have had progressive disease (lymph node enlargement) within one week post therapy. Pharmacokinetic data obtained at the end of infusion, 2, 24 hours and 1 and 2 weeks post infusion showed minimally detectable levels at doses of 3 and 6 mg/kg. At 12 mg/kg, 2 animals had evidence of persistent antibody levels in serum for 24 hours, followed by return to pre-treatment levels within 6 days. Two dogs currently treated at 24 mg/kg maintained hL243 levels in their blood over 1 day, with residual antibody still found in the blood 6 days later Four of the 13 dogs developed anti-hL243 responses 2 to 4 weeks after treatment initiation. These findings provide a rationale for larger clinical studies to investigate the efficacy of anti-HLA-DR mAbs for both veterinary and human applications. Citation Format: Barbara J. Biller, Rodney Page, Robert Sharkey, David M. Goldenberg. Phase I evaluation of anti-human leukocyte antigen-DR monoclonal antibody therapy in spontaneous canine lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A07.

Canine atopic dermatitis reduces the risk of multicentric B cell lymphoma

CANINE atopic dermatitis (AD) has been defined as ‘a genetically predisposed inflammatory and pruritic allergic skin disease with characteristic clinical features associated with IgE antibodies most commonly directed to environmental...

Foxp3+ T-lymphocytes in the pathogenesis of mycosis fungoides

The pathogenesis of mycosis fungoides (MF) is not fully understood, nevertheless it is believed that the microenvironment of the tumor cells could influence the development of malignant lymphocytes. T-regulatory cells are a specialized subpopulation of T-lymphocytes, which can have an immunosuppressive effect and thus affect the immune response. Goal. The study population of regulatory T-cells in the skin of patients with mycosis fungoides and llaque parapsoriasis. Materials and methods. The content of Foxp3+ and CD3+ cells by immunohistochemical studies of skin biopsy specimens from patients with patch stage mycosis fungoides (10 patients) and plaque parapsoriasis (10 patients). The control group consisted of 16 healthy individuals. Results. Relative expression area Foxp3+ cells in patients with mycosis fungoides patch stage (0.44%) 2.6 times greater than in patients with plaque parapsoriasis (0,14%; p &lt; 0,05). Part the Foxp3+ cells of the total number of T lymphocytes in patients with patch stage mycosis fungoides (5.9%) and plaque parapsoriasis (4.7%) were not significantly different (p &gt; 0,05). Conclusion. In the initial stage of patients with mycosis fungoides determined a statistically significant predominance of the absolute number of T-regulatory cells in the epidermis and in the dermis. Part T-regulatory cells from all of T-lymphocytes in patients with skin patch stage mycosis fungoides and plaque parapsoriasis not statistically significantly different.

124: Immunotolerance-inducing IDO-1 and Th1-cytokines are upregulated in subcutaneous panniculitis-like T-cell lymphoma; signs of an immunosuppressive microenvironment

Aims Subcutaneous panniculitis-like T cell lymphomas represent a rare and difficult to diagnose entity of cutaneous T cell lymphomas. SPTL affects predominantly young adults and presents with multifocal subcutaneous nodules and frequently associated autoimmune features. The pathogenesis of SPTL is not completely understood. The aim of this study is to unravel molecular pathways critical to the pathogenesis for the first time. Methods We analyzed 23 skin samples from 20 newly diagnosed SPTL patients and relevant control samples of adipose and non-malignant panniculitis tissue by using gene expression microarray, quantitative PCR, and two-colour immunohistochemistry. Results Interestingly, indoleamine 2,3-dioxygenase ( IDO-1 ), an immunotolerance-inducing enzyme, was among the most highly overexpressed genes in all comparisons. The expression of Th1-specific cytokines, known to be associated with autoimmune inflammation (i.e. IFNG, CXCR3, CXCL9, CXCL10, CXCL11, and CCL5 ), were also significantly increased. Confirmed with immunohistochemistry, the morphologically malignant lymphocytes expressed CXCR3 and CXCL9. IDO-1 expression was found both in some morphologically malignant lymphocytes rimming the adipocytes and in surrounding CD11c − CD68 − cells but not in CD11c + dendritic cells in the microenvironment. The proportion of FoxP3 + cells in SPTL exceeded that in the panniculitis samples. Conclusions Our results indicate that the upregulation of the tolerogenic IDO-1 together with the upregulation of IFNG, CXCR3 ligands, and CCL5 features SPTL lesions. We anticipate that the IFNG-inducible IDO-1 expression contributes to the formation of an immunosuppressive microenvironment, favourable for the malignant T cells. This study provides a relevant molecular basis for further studies exploring novel therapeutic means for subcutaneous T cell lymphoma.

The Society of Biomolecular Imaging and Informatics First Annual Conference

The Society of Biomolecular Imaging and Informatics First Annual Conference

Targeting PI3-kinase (PI3K), AKT and mTOR axis in lymphoma.

Targeted therapy represents a transformation in oncology, a field that has relied primarily on non-selective cytotoxic therapies. Phosphatidylinositol 3-kinase (PI3K) is a family of ubiquitous signalling molecules involved in a wide variety of cellular processes and likewise, in a broad selection of human cancers. The discovery that the p110-δ form of PI3K is differentially expressed in normal and malignant lymphocytes has led to the development of specific inhibitors that are currently in clinical trials for lymphoma. Downstream effectors of PI3K, including v-akt murine thymoma viral oncogene homolog 1 (AKT; also termed AKT1) and mechanistic target of rapamycin (serine/threonine kinase) (mTOR) are similarly important in lymphoma, and agents targeting these components of the PI3K-AKT-mTOR axis are also underway, although at earlier stages of development. In this review we examine the role of PI3K-AKT-mTOR in normal and malignant lymphocytes, as well as the preclinical and clinical status of a number of inhibitors of this pathway.

The thymus in immunity and in malignancy.

The thymus is an essential organ for the generation of the adaptive immune system. By now, the cellular selection events taking place in ongoing life before sexual maturity have been worked out even at the molecular level, and thus thymic lymphocyte development represents one of the best-studied systems in mammalian development. Because thymic lymphocyte development involves ample proliferation and generation of new cells, it is not astonishing that the thymus also represents an organ where malignancy can develop. In this Masters of Immunology primer, the development of lymphocytes and the role of intracellular Notch 1 and cyclins in lymphocytic malignancy are reviewed, offering new therapeutic possibilities.

Neurolymphomatosis: A case series of clinical manifestations, treatments, and outcomes

BackgroundNeurolymphomatosis (NL) is a rare clinical entity characterized by infiltration of malignant lymphocytes into the peripheral nervous system. We analyzed the clinicoradiological features, treatments, and outcomes in NL patients. MethodsWe identified six patients with NL seen at The University of Texas MD Anderson Cancer Center from 01/2010 to 10/2012. We extracted clinical presentations, imagings, CSF cytology, and electrodiagnostic studies from medical records. One patient had a nerve biopsy. We defined therapy response as clinical improvement of neurological deficits. FindingsThe mean age at onset was 57.1years. Most were predominantly men with non-Hodgkin lymphoma. Positron emission tomography (PET) was positive in five patients. Nerve conduction demonstrated mononeuritis multiplex, plexopathy, demyelination, and axonal polyradiculoneuropathy, whereas electromyography was nonspecific. All patients received systemic chemotherapy, four intrathecal chemotherapy, and three intravenous immunoglobulin, plasma exchange or both. One patient who received intravenous immunoglobulin showed mild neurological improvement. Two patients responded, and the median overall survival was 15months. ConclusionsNL is an increasingly recognized complication of NHL and leukemia. A high clinical suspicion is necessary for correct diagnosis. In the present series, patients with leukemia had mononeuritis multiplex, whereas those with lymphoma had plexopathy. Electrodiagnosis and PET scans were useful diagnostic tools. No factors correlated with poorer prognosis. International collaborative studies will help to better determine the risk factors of NL, response to treatment and outcomes.

CNS Relapsed T-cell Lymphoma in a Young Cat

8개월령의 단모종 집고양이가 감소된 식욕과 활력 저하로 내원하였다. 진단을 위한 영상 검사에서 종격동의 종괴와 종격동 림프절의 비대 소견을 확인할 수 있었다. 이어서 진행한 세침흡인술 검사로, 악성 림프구를 다수 확인할 수 있었으며, 이 고양이는 다발성 림프종 (병기 V-b)로 진단되었다. 치료는 COP 프로토콜을 사용하였으며, 완전 완화를 확인할 수 있었지만, 항암 치료를 시작한 후 314일 째 재발과 함께 중추신경계로 전이된 소견을 확인할 수 있었다. 구조화학 요법을 실시하여, 단기적으로는 임상증상의 큰 개선을 확인할 수 있었지만, 부분완화만이 관찰되었으며, 처음 내원 부터 약 383일 정도 생존하였다. 부검과 조직병리학적 검사를 통해, 다발성의 T 세포 림프종으로 확인하였으며, 뇌에서도 병변을 확인할 수 있었다.