Trials In Lymphoma Research Articles

PRM185 - Latest Trends In Design Of Pivotal Phase Iii Clinical Trials For Peripheral T-Cell Lymphoma

PRM185 - Latest Trends In Design Of Pivotal Phase Iii Clinical Trials For Peripheral T-Cell Lymphoma

Protocol for Care After Lymphoma (CALy) trial: a phase II pilot randomised controlled trial of a lymphoma nurse-led model of survivorship care

IntroductionLymphoma is the sixth most common cancer diagnosed in Australia and internationally. Owing to the aggressive nature of the disease and intensity of treatment, survivors face long-term effects that impact...

A Bayesian adaptive phase 1 design to determine the optimal dose and schedule of an adoptive T-cell therapy in a mixed patient population

We present a novel Bayesian adaptive phase 1 design to determine the optimal dosing regimen for an adoptive T-cell therapy in a mixed patient population. Our design is motivated by a B-cell Non-Hodgkin Lymphoma trial evaluating multiple dosing regimens within multiple disease subtypes. A utility score is calculated from both safety and efficacy utility functions and used to guide dose-escalation decisions. We pool safety data across disease subtypes and use a single dose-toxicity model while sharing efficacy information between disease subtypes using a hierarchical dose-response model. In addition, an adaptive randomization approach is applied to dynamically assign patients to a regimen when more than one regimen is open for enrollment. We illustrate this study design through a simulated trial example, and we investigate the operating characteristics using simulation studies.

Abstract BS3-2: Targeting mitochrondria with FDA-approved antibiotics may be a new therapeutic strategy to eliminate cancer stem cells

Abstract Our recent studies indicate that cancer stem cells (CSCs) have increased mitochondrial mass, which directly reflects an increase in mitochondrial biogenesis and OXPHOS capacity. Thus, we believe that more effective anti-cancer therapy would involve the strategic targeting of CSC mitochondria, to prevent or reverse tumor recurrence, metastasis and drug-resistance. Interestingly, mitochondria are originally derived from aerobic bacteria that were engulfed by eukaryotic cells and adapted over millions of years of evolution. This is known as the 'Endo-symbiotic Theory of Mitochondrial Evolution'. As a consequence, certain antibiotics target mitochondrial protein translation as a manageable side effect. We have recently proposed to harness this side effect and to re-purpose it as a therapeutic effect to target breast CSCs. In accordance with this strategy, we have already experimentally identified 4 to 5 different class of mitochondrial-targeted antibiotics that could be used halt the proliferation and eradicate breast CSCs. These antibiotics included azithromycin and doxycycline, among others. Importantly, these antibiotics showed anti-CSC activity in 12 different cell lines, across 8 different cancer types, that were originally derived from breast, DCIS, ovarian, prostate, pancreatic and lung carcinomas, as well as glioblastoma and melanoma. In this context, doxycycline appeared to be one of the most promising agents, as human clinical trials in MALT lymphoma have already shown very positive results. Similarly, clinical trials with azithromycin in lung cancer patients have also shown positive results. Thus, targeting mitochondrial protein translation, with FDA-approved antibiotics, may be a safe effective strategy for inhibiting mitochondrial function in CSCs, regardless of the tumor type or site of origin. For further information on this topic, please see the following references: Antibiotics that target mitochondria effectively eradicate cancer stem cells, across multiple tumor types: Treating cancer like an infectious disease. Lamb R, Ozsvari B, Lisanti CL, Tanowitz HB, Howell A, Martinez-Outschoorn UE, Sotgia F, Lisanti MP. Oncotarget. 2015 Mar 10;6(7):4569-84. Drug therapy: Can the mitochondrial adverse effects of antibiotics be exploited to target cancer metabolism? Killock D. Nat Rev Clin Oncol. 2015 Apr;12(4):190. High mitochondrial mass identifies a sub-population of stem-like cancer cells that are chemo-resistant. Farnie G, Sotgia F, Lisanti MP. Oncotarget. 2015 Sep 30. [Epub ahead of print] PMID: 26421710 http://www.ncbi.nlm.nih.gov/pubmed/26421710 Mitochondrial mass, a new metabolic biomarker for stem-like cancer cells: Understanding WNT/FGF-driven anabolic signaling. Lamb R, Bonuccelli G, Ozsvari B, Peiris-Pages M, Fiorillo M, Smith DL, Bevilacqua G, Mazzanti CM, McDonnell LA, Naccarato AG, Chiu M, Wynne L, Martinez-Outschoorn UE, Sotgia F, Lisanti MP. Oncotarget. 2015 Sep 28. [Epub ahead of print] PMID: 26421711 http://www.ncbi.nlm.nih.gov/pubmed/26421711 Citation Format: Lisanti MP. Targeting mitochrondria with FDA-approved antibiotics may be a new therapeutic strategy to eliminate cancer stem cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr BS3-2.

SRC-3 Ubiquination, Mediated By Cullin3, Potentiates Gambogic Acid-Induced Apoptosis in Non-Hodgkin's Lymphoma

Steroid receptor coactivator-3 (SRC-3) mediates the transcriptional functions of nuclear receptors and other transcription factors, regulating the cell proliferation, survival and growth. SRC-3 amplification or overexpression, detected in many hormone-dependent or -independent tumors, has been demonstrated its important role in promoting carcinogenesis. It was suggested that development of an effective SRC-3 inhibitor could offer a promising therapeutic strategy.Gambogic acid (GA), the major active ingredient in gamboges, is secreted from Garcinia hanburyi. In recent decades, accumulating evidence has been discovered to elucidate its potent anti-tumor efficacies. In our study, we investigated the effects and molecular mechanisms of GA on non-Hodgkin's lymphoma (NHL) cell lines.GA was demonstrated to induce cell growth inhibition, G1 phase cell cycle arrest and apoptosis in NHL. The IC50 for Daudi or Raji at 24 h was 0.15±0.01 μΜ or 0.79±0.23 μΜ, respectively. In control, the IC50 for Jurkat or normal human lymphocytes was 1.324±0.02 μΜ or 2.54±0.21 μΜ, respectively. After incubation with 0.2 μΜ GA for 12 h, the rate of Daudi cells in G1 phase increased from 33.8±1.83% to 64.4±4.4%, with rate of S phase from 53.0±0.14% to 17.99±3.87%. For Raji cells (0.6 μΜ for 12 h), the rate of cells in G1 phase increased from 44.34±1.55% to 57.07±0.5%, with rate of S phase from 44.75±2.2% to 38.16±0.87%. The G1 phase cell cycle arrest was accompanied by the down-regulation of cyclin D3 and p-Rb and up-regulation of P21 and P27. GA (0.2 μΜ for 24 h) greatly induced apoptosis in Daudi cells (from 6.2±2.8% to 46.8±3.2%), but almost none in Raji cells (from 9.5±0.5% to 9±0.72%). When treated with 0.4 μΜ or 0.6 μΜ GA, the rate of apoptosis in Raji cells increased to 40.5±5.5% or 76.5±4.5%. The apoptosis was further confirmed by evaluating the activation of caspase3 with western blot assay. After GA treatment, cyto C and Apaf-1 increased, and Bcl-2 decreased, all indicating GA induced apoptosis in mitochondrial pathway.The effects of GA on the regulations of five oncoproteins (Bcl-6, c-Myc, NF-κB, SRC-3 and Bcl-2) were measured by western blot analysis. These oncoproteins have been considered to be strongly implicated in B-cell lymphomagenesis. SRC-3, Bcl-2 and c-Myc were down-regulated. NF-κB was down-regulated directly or inhibited via up-regulation of IκB-α. The effects of GA on Bcl-6 were contradictory in Daudi and Raji.Because of the pivotal role of SRC-3 in promoting carcinogenesis, we explored whether the above effects of GA were mainly mediated by the regulation of SRC-3. Western blot analysis showed an obvious down-regulation of SRC-3 while the mRNA levels measured by RT-PCR analysis had no marked decline. SRC-3 protein stability examined by cycloheximide assay showed an accelerated degradation of SRC-3. Since SRC-3 has been reported to be degraded in proteasome pathway, we doubted whether SRC-3 was degraded similarly in response to GA. Cullin3, the component of the ubiquitin E3 ligase for SRC-3, was demonstrated to be up-regulated. The proteasome inhibitor MG132 could weaken GA-induced SRC-3 degradation. SRC-3 was shown to be ubiquitinated with immunoprecipitation method, and the ubiquitinated SRC-3 accumulated in the presence of MG132. All the data suggested that GA induced down-regulation of SRC-3 mainly through ubiquitin-dependent proteasome degradation pathway mediated by cullin3.According to GA-induced apoptosis in five cell lines (Daudi, Raji, Pfeiffer, SU-DHL6 and Jurkat), cells with higher SRC-3 level were observed the higher sensitivity. To figure out the relationship of SRC-3 level and cell sensitivity, stable cell clones expressing lower SRC-3 by lentiviral shRNA transfection was established. We compared the antitumor efficacies of GA in SRC-3 siRNA clones with control clones. After treated with 0.6 μΜ GA for 24 h, the rate of apoptosis in SRC-3 siRNA clones or control clones increased from 14.6±3.3% to 44.3±3.7% or from 16.6±2.24% to 63.6±1.99%, respectively (44.3±3.7% vs. 63.6±1.99%, P<0.05). The result showed SRC-3 knockdown was accompanied with reduction of sensitivity, further confirming SRC-3 as a critical target of GA.Taken together, our results demonstrated the potent antitumor efficacies of GA on NHL and revealed that SRC-3 ubiquitination, mediated by Cullin3, played a critical role in this process, providing the molecular basis for the clinical trial in lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Prevalence and Prognostic Value of BCL2 and MYC Protein Expression within ABC and GCB Subtypes in Patients with Previously Untreated, Diffuse Large B-Cell Lymphoma: Analysis from the Phase III MAIN Study

Introduction. Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL). Although more than half of patients are cured with standard immunochemotherapy (R-CHOP) in first-line therapy, the disease relapses or is refractory to R-CHOP in ~40% of cases, with limited options for second-line treatment. Molecular characteristics, including Cell-of-Origin (COO), BCL2 expression and concomitant BCL2/MYC expression, contribute to differences in outcome for DLBCL patients (Alizadeh, Nature 2000, Iqbal Clin Cancer Res. 2011, Johnson JCO. 2012). Understanding these molecular risk factors is potentially of value to guide treatment decisions and optimize therapy. Here, we used assays validated for formalin-fixed, paraffin-embedded (FFPE) specimens to retrospectively assess the prevalence and prognostic value of BCL2 and MYC in patients from MAIN, a Phase III trial that evaluated bevacizumab plus R-CHOP in frontline, CD20-positive DLBCL (NCT00486759). This analysis will help assess markers relevant for risk assessment and may guide use of new investigational agents in DLBCL. Methods. Tissue microarrays (TMAs) from FFPE tumor samples were evaluated using immunohistochemistry (IHC) for BCL2 (clone 124 DAKO), and MYC (clone Y69 Epitomics). FFPE cell pellets from 26 NHL cell lines were also stained as above. BCL2 staining was scored on a 0-3 intensity scale; the BCL2 cutoff was determined by the expression level that conferred sensitivity ( Results. Baseline tissue samples were available from 230/787 (29%) patients from MAIN, with baseline characteristics and survival similar to the overall ITT population. TMAs were available for IHC evaluation in 184/230 (80%) samples, 88/184 (48%) of which were BCL2 positive. BCL2-positivity rates in various sub-sets were: 58% of IPI high, poor prognostic group vs. 41% of IPI low, good prognostic group, and 64% of ABC vs. 40% of GCB samples (Figure 1). BCL2 expression appeared to associate with adverse PFS in the GCB subtype (HR adjusted for IPI, 3.89; 95% CI: 1.30-11.68, Figure 2a) but not in the ABC subtype (HR adjusted for IPI: 0.81; 95% CI: 0.3-2.2, Figure 2b). BCL2 mRNA expression or BCL2 gene rearrangements by FISH showed no association with PFS. Thirty-two of 174 (18%) samples evaluated for MYC and BCL2 by IHC were DP (95% CI: 13%-25%). PFS was similar in non-DP and DP group (HR adjusted by IPI, 0.77; 95% CI: 0.40-1.48), but the DP group appeared to have inferior OS (HR adjusted by IPI, 0.45; 95% CI: 0.22-0.93). These data should be interpreted cautiously due to the small number of PFS/OS events. BCL2 IHC, BCL2 rearrangements and MYC IHC data were available for 40 ABC and 59 GCB samples. DP by IHC was observed in 10/40 (25%) ABC subtype and 10/59 (17%) GCB subtype samples. BCL2 translocations were observed in 6/40 (15%) of ABC, vs. 19/59 (32%) of GCB samples. Conclusions. Using validated assays developed for FFPE tissue testing, we determined the prevalence and prognostic value of BCL2 in a frontline DLBCL population enrolled in a clinical trial. BCL2 expression appeared to be higher in the poor prognostic groups (high IPI and ABC), and appeared to be an independent prognostic factor in the GCB subtype. BCL2 expression, DP and COO may identify patients with DLBCL who could benefit from BCL2 inhibition; the assays employed here are currently in use to evaluate BCL2 as a predictive biomarker for the BCL2 inhibitor venetoclax in lymphoma trials. Disclosures Punnoose:Genentech, Inc.: Employment. Off Label Use: This abstract reports on biomarker analyses using samples taken from previously untreated patients with DLBCL who received bevacizumab in combination with R-CHOP in the MAIN study. Peale:Roche: Equity Ownership; Genentech, Inc.: Employment. Lei:Roche: Employment. Sandmann:F. Hoffmann-La Roche AG: Employment, Equity Ownership. Bourgon:Roche: Equity Ownership; Genentech, Inc.: Employment. Szafer-Glusman:Genentech, Inc.: Employment. Do:Genentech, Inc.: Employment. Salles:Celgene Corporation; Roche: Speakers Bureau; Celgene Corporation; Roche and Gilead Sciences: Research Funding; Calistoga Pharmaceuticals, Inc.; Celgene Corporation; Genentech, Inc.; Janssen Pharmaceutica Products, L.P.; Roche: Consultancy. Seymour:Roche: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Other: Travel support, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Incyte: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Infinity: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Phebra: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Other: Travel support, Research Funding, Speakers Bureau; Genentech, Inc.: Membership on an entity9s Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Trneny:Roche: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding. Munoz:Roche: Employment, Equity Ownership. Mundt:Roche: Employment.

Quantitative Multiplexed Immunohistochemistry Assays for Exploring CAR Modified T Cells and Checkpoint Inhibitors in Lymphoma Trials

Although there have been compelling advances in the cancer immunotherapy space recently in the form of chimeric antigen receptor (CAR) modified T-cells and checkpoint inhibitors, advanced tools to explore the therapeutic mechanisms of their combination are not adequately developed or widely available. To address this growing need, we developed a robust quantitative fluorescent immunohistochemistry platform using multiplex AQUA (Automated Quantitative Analysis) technology to evaluate checkpoint inhibitor expression, enumerate CAR T cells and determine the interaction between tumor cells and immune cells via novel co-localization algorithms. We explored utility of this method both in preclinical- and clinical model systems. In an immunodeficient mouse model of B-cell lymphoma, we evaluated homing of CAR T cells to malignant B-cells in primary lymphoid organs. We determined the phenotype and functional status of the CAR T cells via multiplex analyses of CD4, CD8, PD1 and FOXP3 expression. Additionally, to enable combination immunotherapies in Diffuse Large B-Cell Lymphoma (DLBCL) setting, we explored prevalence of adaptive immune resistance mechanisms in the form of PD1 and PD-L1 expression in immune- and tumor cell compartments via landmarks created by cytoplasmic and nuclear stains in both primary and secondary biopsies from DLBCL patients (n = 63). To support patient selection for CAR T trials, we quantified expression and prevalence of relevant tumor antigens that could not be scored reproducibly by traditional methods to yield objective cut points. We anticipate utilization of these quantitative multiplexed IHC methods for optimal selection of patients into upcoming novel combination immunotherapy trials DisclosuresTran:Genoptix: Employment. Scott:Genoptix: Employment. Lee:Genoptix: Employment. Singh:Novartis: Employment. Cogan:Novartis: Employment. Bordeaux:Genoptix: Employment. Jennifer:Genoptix: Employment. Lameh:Genoptix: Employment. Tribouley:Novartis: Employment. Kassim:Novartis: Employment. Tangri:Genoptix Inc., a Novartis company: Employment. Dakappagari:Genoptix Inc., a Novartis company: Employment.

The Eµ-Myc/TCL1 Transgenic Mouse As a New Aggressive B-Cell Malignancy Model Suitable for Preclinical Therapeutics Testing

Background: Aggressive B-cell lymphomas occurring in the setting of Chronic Lymphocytic Leukemia (CLL) as a large cell transformation are an important clinical problem, and improved mouse models to test novel and targeted therapeutics are needed. The Eµ-Myc mouse overexpresses c-Myc gene which is placed under control of the Myc promoter and lymphoid-specific IgH enhancer (Eµ), resulting in c-Myc overexpression and spontaneous B-cell lymphoma development. The Eµ-Myc mice have been used in drug development, however malignancy develops at variable ages and with differing genetics and response to therapeutic agents, making drug studies difficult. The Eµ-TCL1 transgenic mouse overexpresses the human TCL1 oncogene, under the control of the B-cell specific IgVH promoter and Eµ enhancer. Mice develop a spontaneous mature B-cell leukemia after a long latency period and represent a well-established model of human CLL. We crossed the Eµ-Myc and TCL1 mice to create a new model of aggressive B-cell lymphoma to test novel therapeutics that would be more homogeneous than the Eµ-Myc model.Methods: Eµ-Myc and TCL1 mice on C57BL/6 background strain were crossed and resulting genotypes were verified by PCR. All mice were kept in standard pathogen-free housing until death or removal from study. All mice born within a 24 month time period were followed for survival. At the time of death, spleen, lymph nodes, bone marrow, and liver were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4uM, and stained with hematoxylin and eosin for histologic evaluation. For engraftment studies, adoptive cell transfer was done by injecting fresh splenic white blood cells (1e7 cells, >75% CD5+/CD19+) into the tail vein of C57BL/6 wild type recipient mice. In the drug treatment experiments ibrutinib (~30mg/kg/day) was administered in drinking water and KPT-8602 (15mg/kg) by daily oral gavage. Survival was shown using Kaplan-Meier curves and survival curves compared statistically using a log-rank test.Results: The Eµ-Myc/TCL1 mice had significantly worse survival (median 45.5, range 29-74 days) compared to Eµ-Myc (median 118, range 42-520 days) or TCL1 (median 359.5, range 188-531 days) mice (p=<0.0001). Survival curves are show in Figure 1. Pathologic examination revealed aggressive B-cell histology similar to Burkitt lymphoma in both the Eµ-Myc mice and Eµ-Myc/TCL1 mice, whereas the TCL1 mice showed mature B-cell morphology. Infiltration of the bone marrow of Eµ-Myc and Eµ-Myc/TCL1 mice was extensive in comparison with TCL1 mice. In all three mouse genotypes the normal architecture in the spleen was effaced by neoplastic cells. Adoptive transfer of Eµ-Myc/TCL1 splenic lymphocytes produced similar pathologic findings to the Eµ-Myc/TCL1 transgenic mice with less severe splenic enlargement in the engrafted mice. Median survival of the engrafted mice was short (38 days). There was a differential response to targeted therapeutics between the groups. Ibrutinib, an irreversible inhibitor of Bruton tyrosine kinase (BTK) previously demonstrated to improve survival in TCL1 mice, had no survival advantage over vehicle treatment in either Eµ-Myc or Eµ-Myc/TCL1 mice (p=0.876 and P=0.83 respectively). KPT-8602, a second generation selective inhibitor of exportin 1 (XPO1) was tested in Eµ-Myc/TCL1 engrafted mice, and demonstrated a significant improvement in survival compared to vehicle alone (p=<0.0001). A first generation agent (selinexor) targeting XPO1 is currently in human clinical trials for aggressive lymphomas, including large cell transformation of CLL.Conclusions: Mice with both c-Myc and TCL1 transgenes develop an aggressive B-cell malignancy and have decreased survival compared to Eµ-Myc or TCL1 mice. The histology of the disease is similar to that of Eµ-Myc mice, but with shorter survival with less variability. The resulting mouse model has differential responses to targeted therapeutics, with a poor response to ibrutinib more similar to aggressive lymphoma than CLL. The malignant B cells can be adoptively transferred into immunocompetent mice for experimental drug treatment studies. These qualities make Eµ-Myc/TCL1 mice a useful tool to test new therapies for aggressive lymphoma where there is an unmet clinical need. Work is currently underway to further define the genetic characteristics of this mouse in comparison to human lymphoma sub-types and responsiveness to targeted agents. [Display omitted] DisclosuresByrd:Acerta Pharma BV: Research Funding.

Association of Gene Expression Profiling, Interim PET, EBV Status and PD-L1 Expression in Classical Hodgkin Lymphoma

IntroductionA challenge in the management of classical Hodgkin lymphoma (CHL) is selecting treatment which will maximise success while minimising treatment side effects. The international Response Adapted Therapy in Hodgkin Lymphoma (RATHL) trial (NCT00678327) demonstrated that treatment may be successfully adapted based on positron emission tomography (PET-CT) scan results after 2 cycles (PET2) of ABVD chemotherapy in advanced CHL. This approach however delays risk stratification meaning some patients may be over or under treated and therefore a baseline predictor is highly desirable. A gene expression-based model performed on RNA from formalin-fixed paraffin-embedded tissue (FFPET) biopsies on the Nanostring platform (Scott et al, J Clin Oncol 2013; 31:692-700) has been published. In addition Steidl et al used gene expression profiling (GEP) to correlate tumour-associated macrophages with survival in CHL (Steidl et al, NEJM 2010; 10:875-885). In order to explore whether GEP at baseline could be used in combination with PET2 results to predict outcome, these published gene signatures were explored on the DASL platform in a series of CHL cases and correlated with PET2 response. Recently, PD-1 ligand (PD-L1) on the cell surface of Reed-Sternberg cells (the hallmark cells of CHL) has been demonstrated to co-opt the PD-1 pathway allowing immune evasion (Green et al, Blood 2010;116:3268-77). Therefore this was also performed by immunohistochemistry (IHC) and GEP as well as assessment of EBV status.Methods50 patients with CHL were identified, with at least 2 years of follow up, diagnosed between 2008 and 2013. Deauville scores at PET2 were determined. IHC consistent with CHL, including: CD3, CD19, CD20, CD30, CD79, BOB-1, OCT-2, MUM1 and TARC was performed on 3mm FFPET lymph node sections, and reported by two independent observers. Latent Epstein-Barr virus (EBV) infection was determined by IHC for LMP-1 expression and in-situ hybridisation using fluorescein-labelled peptide nucleic acid probes (Dako, K5201). PD-L1 expression was determined by IHC using a rabbit monoclonal antibody (Cell Signaling Technology, E1L3N, #13684). Three to five 5mm sections of FFPET were used for RNA extraction using the Ambion RecoverAll™ kit standard protocol. GEP was quantified using Illumina's whole genome cDNA-mediated annealing, selection, extension, and ligation Assay (WG-DASL). 61 genes of interest were analysed for significance in the difference in gene expression between groups and included genes from the two recently reported GEP predictor tools in CHL (Steidl et al, 2010, Scott et al 2013) as well as PD-1 and PD-L1. The Mann-Whitney test was used to assess the significance in the difference between means (significant if p<0.05).ResultsA statistically significant difference in EBV status was found between PET negative (Deauville scores 1-3) and PET positive (Deauville scores 4-5) groups (Fisher's exact test: P-Value = 0.028). All patients who were EBV positive had a negative PET2 scan. GEP using WG-DASL revealed that only 2 genes of those reported in recent predictor models (Steidl et al 2010, Scott et al 2013) were expressed at significantly different levels between PET negative and PET positive patients (GLUL, RNF144B), both with increased expression in the PET2 positive group. There was no significant difference between PD-L1 expression and PET scores, CD274 gene expression and outcome. Assessment as to whether samples would cluster into groups according to PET positivity or PD-L1 expression status by unsupervised cluster analysis using four recently reported B cell lymphoma gene signatures (Monti et al, Blood 2005; 5:1851-61, Care et al, PloS one 2013; 2: e55895) was performed, but no significant grouping was found.ConclusionGEP on the DASL platform was not able to predict PET response. The original models were trained on outcome and therefore retraining of the GEP model based on PET response is likely to be required. EBV status was found to be predictive of PET response but this could not be correlated with other biomarkers to predict outcome. Although PD-1 and PD-L1 targeted therapy has shown exciting results in patients with CHL, PD-L1 IHC expression and CD274 gene expression, did not correlate with PET2 response or outcome. Baseline biomarkers capable of identifying patients likely to benefit from targeted treatment needs to be further investigated in proposed clinical trials. DisclosuresNo relevant conflicts of interest to declare.

CC-122 Repairs T Cell Activation in Chronic Lymphocytic Leukemia That Results in a Concomitant Increase in PD-1:PD-L1 and CTLA-4 Immune Checkpoint Expression at the Immunological Synapse

Immunomodulatory drugs (IMiDs®) such as lenalidomide and immune checkpoint blockade (ICB) antibodies can enhance autologous anti-tumor T cell immunity and have the potential to elicit durable control of disease in B cell malignancies. These immunotherapies are likely to be most effective when employed in treatment combinations. Thus, the goal of pre-clinical research should be to reveal mechanisms of action (MOA) in the tumor microenvironment (TME) and identify biomarkers to guide development of combination therapy for patients. CC-122 is a novel first-in-class pleiotropic pathway modifier (PPM®) that has potent anti-proliferative, anti-angiogenic and immunomodulatory activities and is currently in Phase I clinical trials for lymphoma and chronic lymphocytic leukemia (CLL). Here, we have utilized the immunological synapse bioassay to examine T cell interactions with CLL tumor cells (modeling anti-tumor T cell responses in the TME) following CC-122 treatment and measure the expression of co-signaling complexes at the synapse.Conjugation assays and confocal imaging were used to visualize intercellular conjugate interactions and F-actin polymerization at the immune synapse between CD4+ and CD8+ T cells and autologous CLL tumor cells pulsed with superantigen (acting as antigen-presenting cells, APCs). Peripheral blood was obtained from treatment naive CLL patients (n=40) representative of disease heterogeneity. Treatment of both purified CLL cells and CD4+ or CD8+ T cells with CC-122 (0.01 - 1 μM for 24h) dramatically enhanced the number of T cells recognizing tumor cells (% conjugation) and increased the formation of F-actin immune synapses (area, μm2) compared to vehicle treated cells (P<.01). Notably, CC-122 treatment induced T cells to engage in multiple tumor cell synapse interactions that were more pronounced in restored CD8+ T cell lytic synapses. This immunomodulatory activity was detected across all CLL patient samples and drug concentrations tested. In addition, synapse strength as measured by total fluorescence intensity of F-actin per T cell:APC conjugate increased significantly with CC-122 (P<.01). A critical MOA of lenalidomide is activation of T cell immune synapse signaling. Here, our comparative studies revealed that CC-122 (0.1 - 1 μM) significantly enhanced autologous T cell synapse activity in CLL by 4 - 5 fold versus vehicle (P<.01), whereas lenalidomide (1 μM) enhanced activity by 3 fold vs vehicle. Moreover, CC-122 treatment resulted in increased expression and polarization of tyrosine-phosphorylated proteins at T cell synapses compared to lenalidomide and vehicle treatment (P<.01). This data provides evidence that CC-122 induces functional T cell synapses that control the assembly of signaling complexes between the T cell receptor (TCR) and the F-actin cytoskeletal layer. Following T cell recognition of APCs, co-signaling receptors co-localize at the immune synapse where they synergize with TCR signaling to promote (co-stimulatory receptors) or inhibit (co-inhibitory/'immune checkpoint' receptors) T cell activation. Quantitative image analysis studies revealed that restoration of T cell synapse activity with CC-122 was accompanied by an increased recruitment of inducible co-stimulator (ICOS) to the synapse that was dose-dependent (P<.01). CC-122 treatment also increased polarized expression of CTLA-4 and PD-1 immune checkpoint proteins at the synapse with PD-L1+ tumor cells. The observed up-regulation of co-inhibitory receptors led to combining CC-122 with anti-PD-L1, anti-PD-1 or anti-CTLA-4 blocking antibodies. Results show that these treatment combinations increased T cell synapse activity compared to using these immunotherapies alone (P<.01).In conclusion, our results demonstrate for the first time that CC-122 can activate T cell immune synapse signaling against autologous CLL tumor cells and this immunomodulatory capability is more potent than lenalidomide. We further show that CC-122 activation of T cells is associated with enhanced expression of the co-stimulatory receptor ICOS and co-inhibitory checkpoints CTLA-4 and PD-1 at the synapse site. Importantly, our pre-clinical data demonstrates that this regulatory feedback inhibition can be exploited by the addition of anti-PD-L1, anti-PD-1 or anti-CTLA-4 ICB to CC-122 to more optimally stimulate T cell activity against immunosuppressive tumor cells. DisclosuresHagner:Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment. Gandhi:Celgene: Employment, Equity Ownership. Ramsay:MedImmune: Research Funding; Celgene: Research Funding.

Olaparib Recommendations for Ovarian Cancer Patients

Peter Johnson speaks to Gemma Westcott, Commissioning Editor: Peter Johnson is Professor of Medical Oncology at the University of Southampton and Chief Clinician for Cancer Research UK. He graduated from Cambridge University and St Thomas's Medical School (UK). He trained in oncology at St Bartholomew's Hospital, London, where he was an Imperial Cancer Research Fund (ICRF) Clinical Research Fellow and completed his doctoral research on the Bcl-2 gene, its potential as a therapeutic target in lymphoma and the effects of CD40 ligation on the B-cell surface. He was subsequently a Senior Lecturer in Medical Oncology in the ICRF Cancer Medicine Research Unit, Leeds and took up the Chair of Medical Oncology in Southampton (UK) in 1998. He is responsible for bringing together a broad multidisciplinary group of basic, translational and clinical researchers, and linking the research of the academic unit to the extensive clinical practice in cancer treatment in the Southampton Cancer Centre. His research interests are in applied immunology and immunotherapy, lymphoma biology and clinical trials. He is Chief Investigator for lymphoma trials ranging from first in man novel antibody therapeutics to international randomized studies, and for the Cancer Research UK Stratified Medicine Programme. He was Chair of the UK National Cancer Research Institute Lymphoma Group from 2005 to 2011 and has been a member of national trials committees for the Medical Research Council, Cancer Research UK and Leukaemia and Lymphoma Research.

Characterization and pharmacologic targeting of EZH2, a fetal retinal protein and epigenetic regulator, in human retinoblastoma

Retinoblastoma (RB) is the most common primary intraocular cancer in children, and the third most common cancer overall in infants. No molecular-targeted therapy for this lethal tumor exists. Since the tumor suppressor RB1, whose genetic inactivation underlies RB, is upstream of the epigenetic regulator EZH2, a pharmacologic target for many solid tumors, we reasoned that EZH2 might regulate human RB tumorigenesis. Histologic and immunohistochemical analyses were performed using an EZH2 antibody in sections from 43 samples of primary, formalin-fixed, paraffin-embedded human RB tissue, cryopreserved mouse retina, and in whole cell lysates from human RB cell lines (Y79 and WERI-Rb1), primary human fetal retinal pigment epithelium (RPE) and fetal and adult retina, mouse retina and embryonic stem (ES) cells. Although enriched during fetal human retinal development, EZH2 protein was not present in the normal postnatal retina. However, EZH2 was detected in all 43 analyzed human RB specimens, indicating that EZH2 is a fetal protein expressed in postnatal human RB. EZH2 expression marked single RB cell invasion into the optic nerve, a site of invasion whose involvement may influence the decision for systemic chemotherapy. To assess the role of EZH2 in RB cell survival, human RB and primary RPE cells were treated with two EZH2 inhibitors (EZH2i), GSK126 and SAH-EZH2 (SAH). EZH2i impaired intracellular adenosine triphosphate (ATP) production, an indicator of cell viability, in a time and dose-dependent manner, but did not affect primary human fetal RPE. Thus, aberrant expression of a histone methyltransferase protein is a feature of human RB. This is the first time this mechanism has been implicated for an eye, adnexal, or orbital tumor. The specificity of EZH2i toward human RB cells, but not RPE, warrants further in vivo testing in animal models of RB, especially those EZH2i currently in clinical trials for solid tumors and lymphoma.

Additional plerixafor to granulocyte colony-stimulating factors for haematopoietic stem cell mobilisation for autologous transplantation in people with malignant lymphoma or multiple myeloma

Autologous stem cell transplantation is widely used to restore functioning bone marrow in people with malignant lymphoma or multiple myeloma after myeloablative chemotherapy. Results of some clinical trials indicate that plerixafor in addition to granulocyte colony-stimulating factors (G-CSF) compared to G-CSF only could lead to an increased mobilisation and release of CD34-positive cells, facilitating effective apheresis. To evaluate the efficacy and safety of additional plerixafor to G-CSF for haematopoietic stem cell mobilisation in people with malignant lymphoma or multiple myeloma. We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE (from 1990 to September 2015), as well as conference proceedings (American Society of Hematology; American Society of Clinical Oncology; European Hematology Association; American Society for Blood and Marrow Transplantation; European Group for Blood and Marrow Transplantation) for studies. Two review authors independently screened search results. We included randomised controlled trials (RCTs) comparing plerixafor in addition to G-CSF compared to G-CSF only for stem cell mobilisation in people with malignant lymphoma or multiple myeloma of all stages and ages. We included full text as well as abstracts and unpublished data if sufficient information on study design, participant characteristics, interventions, and outcomes was available. We excluded cross-over trials, quasi-randomised trials, and post-hoc retrospective trials. Two review authors independently screened the results of the search strategies, extracted data, assessed quality, and analysed data according to standard Cochrane methods. We performed final interpretation with an experienced clinician. We identified four RCTs fitting the inclusion criteria. However, two of these closed prematurely due to low recruitment and did not report results. The remaining two trials evaluated 600 participants with multiple myeloma or non-Hodgkin lymphoma. In both studies the experimental group received G-CSF plus plerixafor and the control group received G-CSF plus placebo.The meta-analysis showed no evidence for differences between plerixafor and placebo group regarding mortality at 12 months (600 participants; risk ratio (RR) 1.00, 95% confidence interval (CI) 0.59 to 1.69; P = 1.00; moderate-quality evidence) and adverse events during stem cell mobilisation and collection (593 participants; RR 1.02, 95% CI 0.99 to 1.06; P = 0.19; high-quality evidence).Regarding the outcome successful stem cell collection, the meta-analysis showed an advantage for those participants randomised to the plerixafor group (600 participants; RR 2.42, 95% CI 1.98 to 2.96; P < 0.00001; high-quality evidence).As there was high heterogeneity between studies for the number of transplanted participants, we did not meta-analyse these data. In the multiple myeloma study, 95.9% (142 participants) in the plerixafor arm and 88.3% (136 participants) in the placebo arm underwent transplantation (RR 1.09, 95% CI 1.02 to 1.16); in the non-Hodgkin lymphoma trial, 90% (135 participants) in the plerixafor group versus 55.4% (82 participants) in the placebo group could be transplanted (RR 1.62, 95% CI 1.39 to 1.89). In both trials there was no evidence for a difference between participants in the plerixafor and placebo group in terms of time to neutrophil and platelet engraftment in transplanted participants.None of the trials reported on the outcomes quality of life and progression-free survival. The results of the analysed data suggest that additional plerixafor leads to increased stem cell collection in a shorter time. There was insufficient evidence to determine whether additional plerixafor affects survival or adverse events.The two trials included in the meta-analysis, both of which were conducted by the Genzyme Corporation, the manufacturer of plerixafor, were published several times. Two more RCTs examining the addition of plerixafor to a G-CSF mobilisation regimen terminated early without publishing any outcome. The trials included nine and five participants, respectively. Another RCT with 100 participants was recently completed, but has not yet published outcomes. Due to the unpublished RCTs, it is possible that our review is affected by publication bias, even though two trials failed to recruit a sufficient number of participants to analyse any data.

The exclusion of people living with HIV (PLWH) from clinical trials in lymphoma.

Background:The prognosis of non-Hodgkin lymphoma and Hodgkin lymphoma is not affected by HIV serostatus, yet people living with HIV (PLWH) are frequently excluded from clinical trials in lymphoma.Methods:The UK NIHR Clinical Research Network Study Portfolio website was used to identify all the open clinical trials in lymphoma in the United Kingdom in January 2015. Trials that excluded PLWH were further investigated to evaluate if the exclusion was justified by scientific evidence.Results:We identified 56 multicentre open clinical trials in lymphoma including 46 interventional trials. People living with HIV were excluded from 32 interventional trials (70%). We identified a biologically valid reason (a potential increased risk of greater immunosuppression) for excluding PLWH from one trial and possibly for one optional arm in another study.Conclusions:There was no scientific or safety justification for excluding PLWH from most lymphoma clinical trials included on the NIHR portfolio. A clear justification for excluding PLWH was not offered in the available protocols. The exclusion of PLWH should be explicitly justified on scientific grounds in protocols to minimise stigmatisation.

Abstract 1332: Interleukin-15 enhances rituximab-dependent cytotoxicity ex vivo and in vivo against a mouse lymphoma expressing human CD20

Abstract Rituximab (RTX), an anti-CD20 antibody, revolutionized treatment for B-cell malignancies, but it is not without its own shortcomings, most notably tumor relapse. Recent research has provided evidence supporting the increased efficacy of RTX when combined with interleukin-15 (IL-15). IL-15 enhances antibody-dependent cellular cytotoxicity (ADCC), an important mechanism of RTX, by increasing the proliferation and activation of natural killer (NK) cells, as well as monocytes and macrophages. However, the majority of this evidence has been obtained through in vitro experiments and in vivo models using xenografts in immuno-deficient mice. Given the complexity of the immune system, we used an immuno-competent, syngeneic mouse model of human B-cell lymphoma to further investigate the effect of combining IL-15 with RTX to enhance ADCC. Wild-type (WT) C57BL/6 mice (n = 40) were distributed into treatment groups of 10 mice each, and inoculated intravenously with EL4-CD20 cells, a mouse lymphoma line transfected with human CD20. IL-15 was given five times per week for 4 weeks (5μg/mouse), starting on day 3 after tumor inoculation, and RTX was given once per week for 4 weeks (100 μg/mouse), starting on day 5. While IL-15 and RTX individually prolonged survival of the mice when compared with the control (p&amp;lt;0.05), the combination of IL-15 and RTX showed an additive effect, prolonging mouse survival significantly when compared with the monotherapies alone (p&amp;lt;0.01). At day 75 after tumor inoculation, 90% of the mice in the combination group were still alive. In comparison, only 30% of the mice in each of the IL-15 and RTX groups and none of the mice in the control group were alive. In contrast, treatment of EL4-CD20-bearing FcRγ-/- mice with RTX, under the same dose and dosing schedule as those used in WT mice, did not show a therapeutic effect when compared with the control. We further confirmed the role of ADCC by performing an ex vivo experiment using NK cells purified from splenocytes of either WT or FcRγ-/- mice treated with IL-15 five days before extraction. NK cells were cultured in triplicate with 51Cr-labeled EL4-CD20 cells with or without RTX and lysis was evaluated using a chromium-51 release assay. NK cells from IL-15-treated WT mice combined with RTX resulted in a three-fold increase in ADCC when compared with the monotherapies alone (16% combination vs. 6% IL-15 and 5% RTX, at a 10:1 E:T ratio). In contrast, NK cells from FcRγ-/- mice showed no difference in ADCC with or without RTX treatment. In agreement with previously published research, our study gives further evidence that IL-15 increases the efficacy of RTX, primarily through enhancing ADCC. Confirming this synergistic effect in an immuno-competent mouse model better supports a future study of RTX with IL-15 in human clinical trials for indolent B-cell lymphomas. Citation Format: Bernard Wen, Meili Zhang, David Dilillo, Jeffrey V. Ravetch, Thomas A. Waldmann. Interleukin-15 enhances rituximab-dependent cytotoxicity ex vivo and in vivo against a mouse lymphoma expressing human CD20. [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 1332. doi:10.1158/1538-7445.AM2015-1332

Abstract 208: Novel intracellular Doppler imaging predicts therapeutic efficacy for personalized medicine

Abstract This study reports first application of biodynamic imaging (BDI), a novel Doppler imaging technology, in studies of ovarian cancer xenografts and preclinical trials of canine multicentric lymphoma responding to doxorubicin. In a study of A2780 human ovarian cell lines implanted in mice, an unexpected and large difference was discovered between mouse tumor explants versus A2780 cells grown as 3D tumor spheroids. The biopsied explants from mice were three times more sensitive to platinum, applied in vitro, than were multicellular tumor spheroids. A preclinical trial has been completed for canine non-Hodgkins lymphoma using BDI to predict patient outcome under doxorubicin therapy. The 3D imaging capability of BDI enabled the measurement of tumor heterogeneity in response to the therapy. The biodynamic assay took only 24 hours to complete, while the clinical outcome was often unknown for one to several months. The therapeutic efficacy of doxorubicin was predicted by BDI with 90% accuracy. Based on this success, biodynamic imaging is entering human pilot trials in ovarian, pancreatic, and esophageal cancer. Biodynamic imaging is a radically different approach to the prediction of therapeutic efficacy for personalized medicine with higher accuracy and more biological relevance than standard cell-based assays. Supported in part by NIH NIBIB 1R01EB016582-01. Citation Format: John J. Turek, Dan Merrill, Michelle Custead, Michael Childress, David D. Nolte, Bakhtiyor Yakubov, Ran An, Daniela Matei. Novel intracellular Doppler imaging predicts therapeutic efficacy for personalized medicine. [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 208. doi:10.1158/1538-7445.AM2015-208

FDG-PET for the early treatment monitoring, for final response and follow-up evaluation in lymphoma

Optimal lymphoma management requires reliable assessment of response, both during and after therapy. Positron emission tomography with computerized tomography (PET/CT) combines functional and anatomical imaging and provides the most sensitive and accurate method for lymphoma imaging. New guidelines for lymphoma imaging and recently revised criteria for lymphoma staging and response assessment recommend PET/CT for both staging, treatment monitoring and response evaluation in all 2-[18F]fluor-2-deoxyglucose (FDG)-avid lymphomas, while CT remains the method of choice for non-FDG-avid histologies. Since interim PET imaging has high prognostic value in lymphoma, a number of trials investigate PET-based, response-adapted lymphoma therapy. PET response is the main determinant of final treatment evaluation according to the new response criteria but PET/CT has little or no role in routine surveillance imaging, which has questionable value in general. This review presents from a clinical point of view the evidence for the use of PET/CT in malignant lymphoma during and after therapy. The reader is given an overview of the current PET-based interventional Hodgkin and non-Hodgkin lymphoma trials and an insight into possible future developments in the field, including new PET tracers.

PET response-adapted clinical trials in Hodgkin lymphoma: a comprehensive review

Hodgkin lymphoma (HL) is a highly curable disease, as most HL patients become long-term survivors, with 10-years cure and survival rates after first-line treatment exceeding 80 and 90 %, respectively. Despite these rewarding results, 10–15 % of early stage and 20–25 % of advanced stage patients became chemo-refractory to first-line treatment, and nearly half of them ultimately succumb to their disease. On the other hand, a small but significant fraction of long-term survivors, especially those treated with combined chemoradiation, experience treatment related morbidity and mortality with cardiovascular events and secondary neoplasms arising 5–20 years after the end of therapy. Therefore, an unfulfilled need still exists for a risk-adapted strategy, with a very effective treatment in high-risk patients and a low toxicity regimen in the vast majority of patients who are indeed chemo sensitive. Interim PET scan, performed after few cycles of chemotherapy proved the main predictor of treatment outcome in ABVD-treated patients and superseded the predictive role of traditional prognostic markers. Moreover, simple and reproducible rules for interim PET interpretation agreed among experts have been validated in HL, and are now available for the clinical practice. A number of phase II trials yielded the prof of concept that adapting treatment to interim PET result could ultimately increase long-term disease control in the entire patient population, compared to that obtained with the standard chemotherapy approach. In the present article the results of these pioneer single-centre small trials, as well as the preliminary results of large international prospective trials will be reviewed.

Transformative Clinical Trials in Non-Hodgkin and Hodgkin Lymphomas.

Dramatic progress in the understanding of underlying disease biology and the development of novel therapeutics has yielded a revolution that is poised to transform the face of lymphoma treatment across a broad spectrum of histologies. Ongoing randomized clinical trials are poised to unseat long-entrenched standards of care in diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, peripheral T-cell lymphoma, and Hodgkin lymphoma. Emerging treatment approaches are reviewed, including optimization of existing chemoimmunotherapy platforms, development of chemotherapy-sparing immunotherapy for follicular lymphoma, biologically targeted therapy for subsets of diffuse large B-cell lymphoma, and incorporation of novel agents into the treatment of mantle cell lymphoma and peripheral T-cell lymphoma. Novel therapies in early stage trials with future promise of redefining standards of care are also reviewed for non-Hodgkin and Hodgkin lymphomas, including small molecule pathway inhibitors and advances in immunotherapy.

Non-Hodgkin lymphoma in children with an associated inherited condition: A retrospective analysis of the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP).

Inherited conditions affecting genetic aberration, viral oncogenesis, reduced immune surveillance, and long-lasting antigen stimulation may build the way to lymphomagenesis in humans. We extracted from the database of 4 consecutive trials for pediatric non-Hodgkin lymphoma (NHL) all cases with an associated genetic disease. Among 1,430 patients, 34 (2.4%) had an associated inherited condition and a mature B-lineage (n = 28), anaplastic large cell lymphoma (n = 4), or T-lineage (n = 2) NHL. Their median age at the diagnosis was 9.3 years (range, 2.6-17.8 years). In 14 cases (41%) the underlying condition was considered to be a potential cause for undue toxicity if the expected therapy was applied. Thus, treatment modification had been planned in advance. The overall survival was 89% (standard error [SE] 1%), 73% (SE 10%), and 73% (SE 23%) at 3 years for registered patients with no inherited condition associated, with genetic abnormalities and with underlying condition causing an immune deficiency, respectively (P = 0.003). In our cohort, patients with NHL with an underlying constitutional condition represent the 2.4% of the cases. In the subset of patients with primary immune deficiency, which may have contributed to lymphomagenesis, allogeneic hematopoietic stem cell transplantation may be required. In the remaining patients, the association with lymphoma remains apparently unexplained and could be not causative. Detailed reporting of such cases may contribute to disclose even rare and fully unexpected association, which may have implications for research in the field of lymphomagenesis.